This review discusses label-free cell separation and sorting in microfluidic systems. Cell separation and sorting are essential in cell biology research and many diagnostic and therapeutic methods. Recent interest has focused on methods that avoid biochemical labels, using intrinsic biomarkers such as size, electrical polarizability, and hydrodynamic properties. Microfluidic techniques are used for label-free discrimination and fractionation of cell populations. These techniques are often used to handle single cells and interface with other tools for biochemical analysis. The review highlights recent developments and evaluates their potential for application. It considers inertial effects and proposes general performance metrics for comparison. The current state of these technologies is assessed, and directions for improvement are suggested. The review covers various techniques for cell separation and sorting, including microscale filters, hydrodynamic filtration, deterministic lateral displacement, field-flow fractionation, microstructures, inertial separation, gravity and sedimentation, biomimetic methods, magnetophoresis, aqueous two-phase systems, and acoustophoresis. Each technique is discussed in terms of its principles, performance metrics, and applications. The review emphasizes the importance of intrinsic cellular biomarkers and the potential of microfluidic systems for label-free sorting. It also discusses the challenges and limitations of these techniques, including clogging, fouling, and low throughput. The review concludes that microfluidic systems offer a promising approach for label-free cell separation and sorting, with potential applications in diagnostics, therapeutics, and cell biology.This review discusses label-free cell separation and sorting in microfluidic systems. Cell separation and sorting are essential in cell biology research and many diagnostic and therapeutic methods. Recent interest has focused on methods that avoid biochemical labels, using intrinsic biomarkers such as size, electrical polarizability, and hydrodynamic properties. Microfluidic techniques are used for label-free discrimination and fractionation of cell populations. These techniques are often used to handle single cells and interface with other tools for biochemical analysis. The review highlights recent developments and evaluates their potential for application. It considers inertial effects and proposes general performance metrics for comparison. The current state of these technologies is assessed, and directions for improvement are suggested. The review covers various techniques for cell separation and sorting, including microscale filters, hydrodynamic filtration, deterministic lateral displacement, field-flow fractionation, microstructures, inertial separation, gravity and sedimentation, biomimetic methods, magnetophoresis, aqueous two-phase systems, and acoustophoresis. Each technique is discussed in terms of its principles, performance metrics, and applications. The review emphasizes the importance of intrinsic cellular biomarkers and the potential of microfluidic systems for label-free sorting. It also discusses the challenges and limitations of these techniques, including clogging, fouling, and low throughput. The review concludes that microfluidic systems offer a promising approach for label-free cell separation and sorting, with potential applications in diagnostics, therapeutics, and cell biology.