4'-Carbomethoxyphenyl 4-guanidinobenzoate mesylate (4'-CMGB) is a sperm acrosin inhibitor. A validated reversed- phase isocratic high-performance liquid chromatographic (HPLC) method for its assay and related substances detection is described. The chromatographic system consists of a C(8) Shimpack column (150 mm x 4.6 mm, 5 microm) with a mobile phase composition of methanol-0.2% ammonium acetate-triethylamine-glacial acetic acid (470:524:1:5, v/v/v/v) and UV detection at 266 nm. The method shows good chromatographic separation for 4'-CMGB and its related substances. The method is found to be linear in the range of 2.0-100 microg/mL, precise, and sensitive (limit of detection of 15 ng/mL). The major related substances are resolved and identified by HPLC and LC-tandem mass spectrometry as p-guanidino benzoic acid, methyl p-guanidino benzoate, and methyl p-hydroxybenzoate.