It is a challenge to determine the quantity of cationic finishing agents on the surface of cellulosic fabrics. Herein, we report a direct and feasible method by Kubelka-Munk equation to quantify the cationic poly (hexamethylene biguanide) hydrochloride (PHMB) adsorbed onto cotton fabrics based on the principle of formation of a stable blue dye between PHMB and bromophenol blue sodium (BPB). The adsorption of PHMB onto cotton fabrics was first investigated and the maximum adsorption of PHMB was found to be around 8 mg per gram of cotton fabric. After being dyed with BPB, colour strength shows a positive correlation with PHMB at low concentrations (<2400 mg/L). A linear relationship with a high correlation (C(PHMB) = (K/S-0.7411)/3.4517, R 2 =0.9983) was thus established between colour strength and PHMB concentration. However, this equation should fulfill four requirements for quantifying PHMB: (1) the distribution of PHMB on the surface of cellulosic fabric should be in the form of a monolayer with the content less than 5.3 mg/g; (2) an excess of BPB dyebath should be applied; (3) the dyeing should come to equilibrium; and (4) the fabrics should be evenly dyed. Moreover, MTT assay results indicate that excess PHMB coated on cotton fabrics can cause cytotoxicity and the quantity of PHMB should not exceed 4.62 mg per gram of cotton fabrics for the purpose of biosafety. The sample can be considered noncytotoxic if the K/S value is lower than 13.2 after dyeing with BPB.