Abstract : Using rat serum as a source of esterase activity, we describe a quick and efficient method of taking advantage of the water solubility of the paranitrophenoxide anion. Esterase activity is terminated by adding chloroform and releasing paranitrophenol converted to the 400 nanometers absorbing paranitrophenoxide anion by adding 0.2 molar phosphate buffer, pH (hydrogen-ion activity) 9.0. Paranitrophenoxide is quantitatively partitioned into the aqueous phase, while leaving unhydrolyzed substrate in the organic (chloroform) phase. Partitioned paranitrophenoxide is monitored spectrophotometrically at 400 nm. This method allows quantitation either by direct comparison of identically treated standards or by utilization of appropriate Molar Extinction Coefficients independent of assay pH. Keywords: Esterases, Biochemistry, Colorimetric assay, Chromogenic substrates, Enzymes.