This study presents a microfluidic system that mimics the natural silk spinning process of spiders to replicate the self-assembly of spider silk. The system uses recombinant MaSp2 spidroin and incorporates ion-induced liquid-liquid phase separation, pH-driven fibrillation, and shear-dependent β-sheet formation. The results show that a threshold shear stress of approximately 72 Pa is required for fiber formation, and β-sheet formation is dependent on the presence of polyalanine blocks in the repetitive sequence. The MaSp2 fiber formed has a β-sheet content (29.2%) comparable to that of native dragline silk with a shear stress requirement of 111 Pa. The study also demonstrates the formation of hierarchically structured silk fibers from recombinant MaSp2 precursors with tunable β-sheet abundance and at near-instantaneous speeds. The microfluidic system was designed to approximate the native-like chemical and physical gradients within the channel for the investigation of spider silk fiber assembly. The system uses sequential administration of biomimetic chemical gradients that trigger phase separation and nanofibril formation, and through quantifiable shear effects, it demonstrates the complete in situ assembly of hierarchically structured silk fibers. The study also quantifies shear-induced crystallization and shows that the β-sheet content increases with shear stress, reaching up to 29.2% in section C. The results provide insights into the shear-induced crystallization and sequence-structure relationship of spider silk and have significant implications for the rational design of artificially spun fibers. The study also highlights the importance of shear stress in the self-assembly of spider silk and the potential for tuning fiber structure via subtle changes in the applied pressure. The study also discusses the self-assembly mechanism of spider silk, showing that LLPS is the initial step during silk assembly and is remarkably critical for the following nanofibrillization that is responsible for hierarchical organization. The study also discusses the role of different domains in successful self-assembly of spider silk under native conditions. The study also discusses the use of microfluidics in the assembly of structural proteins and the potential for this technology in the rational design of artificially spun fibers. The study also discusses the use of computational models to understand the MaSp2 fiber assembly mechanism and the role of physical forces in fiber formation. The study also discusses the use of various techniques such as Raman spectroscopy, wide-angle X-ray scattering, and scanning electron microscopy to characterize the β-sheet formation and hierarchical structure of the silk fibers. The study also discusses the preparation of recombinant MaSp2 proteins and the methods used for their purification and characterization. The study also discusses the fabrication of the microfluidic device and the methods used for its operation and maintenance. The study also discusses the use of various imaging techniques such as optical microscopy, confocal laser scanning microscopy, and scanning electron microscopy to characterize the morphology and structure of the silk fibers. The study also discusses the use ofThis study presents a microfluidic system that mimics the natural silk spinning process of spiders to replicate the self-assembly of spider silk. The system uses recombinant MaSp2 spidroin and incorporates ion-induced liquid-liquid phase separation, pH-driven fibrillation, and shear-dependent β-sheet formation. The results show that a threshold shear stress of approximately 72 Pa is required for fiber formation, and β-sheet formation is dependent on the presence of polyalanine blocks in the repetitive sequence. The MaSp2 fiber formed has a β-sheet content (29.2%) comparable to that of native dragline silk with a shear stress requirement of 111 Pa. The study also demonstrates the formation of hierarchically structured silk fibers from recombinant MaSp2 precursors with tunable β-sheet abundance and at near-instantaneous speeds. The microfluidic system was designed to approximate the native-like chemical and physical gradients within the channel for the investigation of spider silk fiber assembly. The system uses sequential administration of biomimetic chemical gradients that trigger phase separation and nanofibril formation, and through quantifiable shear effects, it demonstrates the complete in situ assembly of hierarchically structured silk fibers. The study also quantifies shear-induced crystallization and shows that the β-sheet content increases with shear stress, reaching up to 29.2% in section C. The results provide insights into the shear-induced crystallization and sequence-structure relationship of spider silk and have significant implications for the rational design of artificially spun fibers. The study also highlights the importance of shear stress in the self-assembly of spider silk and the potential for tuning fiber structure via subtle changes in the applied pressure. The study also discusses the self-assembly mechanism of spider silk, showing that LLPS is the initial step during silk assembly and is remarkably critical for the following nanofibrillization that is responsible for hierarchical organization. The study also discusses the role of different domains in successful self-assembly of spider silk under native conditions. The study also discusses the use of microfluidics in the assembly of structural proteins and the potential for this technology in the rational design of artificially spun fibers. The study also discusses the use of computational models to understand the MaSp2 fiber assembly mechanism and the role of physical forces in fiber formation. The study also discusses the use of various techniques such as Raman spectroscopy, wide-angle X-ray scattering, and scanning electron microscopy to characterize the β-sheet formation and hierarchical structure of the silk fibers. The study also discusses the preparation of recombinant MaSp2 proteins and the methods used for their purification and characterization. The study also discusses the fabrication of the microfluidic device and the methods used for its operation and maintenance. The study also discusses the use of various imaging techniques such as optical microscopy, confocal laser scanning microscopy, and scanning electron microscopy to characterize the morphology and structure of the silk fibers. The study also discusses the use of