Extrinsic fluorescent dyes are widely used in protein analysis for characterizing folding intermediates, measuring surface hydrophobicity, and detecting aggregation or fibrillation. These dyes, such as ANS, Bis-ANS, Nile Red, Thioflavin T, and Congo Red, are noncovalent and sensitive to their environment, with fluorescence properties influenced by solvent relaxation, (twisted) intramolecular charge transfer (ICT), and interactions with proteins. Their versatility, sensitivity, and suitability for high-throughput screening make them valuable tools for protein characterization. The review discusses the spectral properties and applications of various extrinsic dyes. ANS and Bis-ANS are sensitive to solvent polarity, viscosity, and temperature, with fluorescence changes indicating hydrophobic interactions with proteins. Nile Red's fluorescence is influenced by solvent polarity and hydrogen bonding, while DCVJ is sensitive to environmental viscosity. Thioflavin T is effective for detecting amyloid fibrils, with fluorescence changes indicating interactions with β-sheet structures. Congo Red is used for amyloid fibril analysis, showing UV absorbance shifts and birefringence in the presence of fibrils. Extrinsic dyes can detect protein aggregation, fibrillation, and conformational changes. They are useful for assessing surface hydrophobicity, monitoring protein interactions with other molecules, and visualizing protein aggregates and crystals. These dyes also provide insights into protein structure, folding intermediates, and aggregation processes without the need for chromatographic separation. Their high sensitivity and versatility make them valuable for early-stage formulation development and high-throughput screening. Extrinsic dyes complement intrinsic fluorescence by providing information independent of aromatic amino acids, making them essential for characterizing hydrophobic proteins and monitoring subtle structural changes. Overall, extrinsic fluorescent dyes are highly sensitive and versatile tools for protein characterization, contributing to the safety and quality of protein pharmaceuticals.Extrinsic fluorescent dyes are widely used in protein analysis for characterizing folding intermediates, measuring surface hydrophobicity, and detecting aggregation or fibrillation. These dyes, such as ANS, Bis-ANS, Nile Red, Thioflavin T, and Congo Red, are noncovalent and sensitive to their environment, with fluorescence properties influenced by solvent relaxation, (twisted) intramolecular charge transfer (ICT), and interactions with proteins. Their versatility, sensitivity, and suitability for high-throughput screening make them valuable tools for protein characterization. The review discusses the spectral properties and applications of various extrinsic dyes. ANS and Bis-ANS are sensitive to solvent polarity, viscosity, and temperature, with fluorescence changes indicating hydrophobic interactions with proteins. Nile Red's fluorescence is influenced by solvent polarity and hydrogen bonding, while DCVJ is sensitive to environmental viscosity. Thioflavin T is effective for detecting amyloid fibrils, with fluorescence changes indicating interactions with β-sheet structures. Congo Red is used for amyloid fibril analysis, showing UV absorbance shifts and birefringence in the presence of fibrils. Extrinsic dyes can detect protein aggregation, fibrillation, and conformational changes. They are useful for assessing surface hydrophobicity, monitoring protein interactions with other molecules, and visualizing protein aggregates and crystals. These dyes also provide insights into protein structure, folding intermediates, and aggregation processes without the need for chromatographic separation. Their high sensitivity and versatility make them valuable for early-stage formulation development and high-throughput screening. Extrinsic dyes complement intrinsic fluorescence by providing information independent of aromatic amino acids, making them essential for characterizing hydrophobic proteins and monitoring subtle structural changes. Overall, extrinsic fluorescent dyes are highly sensitive and versatile tools for protein characterization, contributing to the safety and quality of protein pharmaceuticals.