A new sensitive assay for pyrimidine nucleoside phosphorylase using non-radioactive substrates is described. With the natural substrate uridine (UR) and the analog, 5'-deoxy-5-fluorouridine (5'dFUR) conditions have been optimized to measure the product formation with reversed-phase high-performance liquid chromatography. Using automated injection large series of samples may be analyzed. The assay for UR phosphorylase appeared to be comparable to existing methods with radiolabeled UR as substrate regarding sensitivity and linearity. The assay has been used to measure kinetic parameters for 5'dFUR and UR in two cell lines from intestinal origin.