A method of quantifying an ethylene oxide based nonionic surfactant in a sample containing a physiologically active protein, characterized in that quantitation is performed by colorimetry, in particular, a method of quantifying an ethylene oxide based nonionic surfactant in a sample containing a physiologically active protein, characterized in that an ammonium thiocyanate/cobalt nitrate reagent (CoSCN reagent) is added to said sample solution and the resulting cobalt thiocyanate complex is quantified by absorbance measurement.