A synthetic heptapeptide (H-Pro-Thr-Glu-Phe-[p-nitro-Phe]-Arg-Leu-OH) was used as substrate for detection and assay of cathepsin D in raw bovine milk. Cathepsin D produced a specific peptide, as detected by HPLC analysis of peaks for product and substrate. On incubation of acid wheys from milk samples with the substrate, three hydrolysis products were detected and bonds cleaved were identified by mass spectrometry. Inhibition studies were performed to identify enzymes responsible for the hydrolysis. One activity was cathepsin D and production of another peptide was inhibited by cysteine protease inhibitors, suggesting cysteine protease activity in milk.