4-Nitrophenyl 4-guanidinobenzoate (NPGB) serves as a chromogenic active-site titrant and substrate for serine proteases, especially trypsin and trypsin-like enzymes. It reacts with the active-site serine to form a covalent acyl-enzyme intermediate, releasing 4-nitrophenol that generates a yellow color detectable at 405–410 nm. The initial rapid burst of color release enables precise quantification of active enzyme concentration, while the subsequent slow hydrolysis supports studies of enzyme kinetics and inhibition. Due to its specificity, stability, and sensitivity, it is widely used in biochemical research, diagnostic assays, and high-throughput screening for pharmaceutical applications targeting protease-mediated processes, such as inflammation, blood coagulation, and cancer cell metastasis. It also acts as a mechanism-based inhibitor and lead compound for developing therapeutics against dysregulated proteases.