This review discusses the use of noncovalent, extrinsic fluorescent dyes in protein characterization. These dyes, such as ANS, Bis-ANS, Nile Red, Thioflavin T, and Congo Red, are applied in various fields including characterizing folding intermediates, measuring surface hydrophobicity, and detecting aggregation or fibrillation. The underlying mechanisms of fluorescence properties, such as solvent relaxation and intramolecular charge transfer reactions, are explained. The review highlights the versatility, sensitivity, and suitability of these dyes for high-throughput screening. It provides an overview of the spectral properties and characteristics of commonly used dyes, and illustrates their applications in protein characterization with examples. The review also covers practical considerations for using these dyes and discusses their use in assessing protein denaturation, folding, molten globule intermediates, protein aggregation, amyloid fibril detection, surface hydrophobicity, and protein interactions. Finally, it explores new trends in protein characterization using extrinsic fluorescent dyes, emphasizing their high sensitivity and suitability for high-throughput studies.This review discusses the use of noncovalent, extrinsic fluorescent dyes in protein characterization. These dyes, such as ANS, Bis-ANS, Nile Red, Thioflavin T, and Congo Red, are applied in various fields including characterizing folding intermediates, measuring surface hydrophobicity, and detecting aggregation or fibrillation. The underlying mechanisms of fluorescence properties, such as solvent relaxation and intramolecular charge transfer reactions, are explained. The review highlights the versatility, sensitivity, and suitability of these dyes for high-throughput screening. It provides an overview of the spectral properties and characteristics of commonly used dyes, and illustrates their applications in protein characterization with examples. The review also covers practical considerations for using these dyes and discusses their use in assessing protein denaturation, folding, molten globule intermediates, protein aggregation, amyloid fibril detection, surface hydrophobicity, and protein interactions. Finally, it explores new trends in protein characterization using extrinsic fluorescent dyes, emphasizing their high sensitivity and suitability for high-throughput studies.