ABSTRACTA semi‐automated colorimetric method to measure aminopeptidase activity using reflectance colorimetry is described. p‐Nitroanilide derivatives of 12 amino acids were used to detect aminopeptidase activity in phosphate buffer and milk. Enzyme activity, in phosphate buffer, determined using spectrophotometry and colorimetry, was linearly correlated (R2= 0.991), indicating colorimetry can be used in place of spectrophotometry to measure aminopeptidase activity. Aminopeptidase activity determined colorimetrically in buffer was linearly correlated (R2= 0.981) with activity in milk, indicating colorimetry can be used to detect enzyme activity in turbid solutions such as milk and dairy products. Optimum substrate concentration differed for milk and phosphate buffer. Assays in milk required higher concentrations of chromogenic substrates (0.8–1.0 mM), than did assays in phosphate buffer (0.3–0.5 mM). The assay was more repeatable in milk (average coefficient of variation = 3.6%) than in buffer (average coefficient of variation = 14.0%). Aminopeptidase activity of cell free extracts from Lactobacillus helveticus CNRZ32 were used to demonstrate the use of the assay. Enzyme profiles of this strain were similar in milk and in phosphate buffer. High activity was detected with p‐nitroanilide derivatives of arginine, lysine, leucine, alanine, and m