We describe a method to determine the substrate specificity of human lysosomal carboxypeptidase, cathepsin A/protective protein, using furylacryloyl (FA)-Phe-X dipeptides as substrates. These dipeptides contain a chromophore which allows continuous spectrophotometric assay at wavelengths above 324 nm with little interference from protein absorbance. The results obtained with cathepsin A purified from human placenta demonstrate that the enzyme has the highest affinity for substrates with large hydrophobic (Phe, Leu) or positively charged (Arg) amino acid residues in P1' position. The three substrates (FA-Phe-Phe, FA-Phe-Leu, and FA-Phe-Ala) which demonstrated the highest specificity (kcat/Km) for the purified enzyme were then used to assay cathepsin A activity in cultured skin fibroblasts from patients affected with galactosialidosis, an inherited lysosomal storage disease caused by the genetic deficiency of cathepsin A. Residual cathepsin A activity in galactosialidosis fibroblasts was lower than 6% of controls, indicating the high specificity of the assay method.