We have investigated the correlation between urinary isatin concentration and MAO-B inhibitory activity. A sensitive enzyme-linked immunosorbent assay (ELISA) for the measurement of isatin in urine was developed. There was a strong correlation between isatin concentrations determined by this method and the established GC/MS technique. In two sets of urine samples from healthy individuals (N = 63 and N = 21), we have shown that there is no significant correlation between isatin concentration and MAO-B inhibitory activity (r = − 0.1013 and − 0.1597, respectively). Based upon IC50 measures, the concentration of isatin in normal urine (N = 10) was 3– 5 times less than that which could account for the detected MAO-B inhibitory activity. Furthermore, Lineweaver-Burk plots from urine samples (N = 12) compared with isatin showed that the inhibition by isatin was competitive whereas urine samples could elicit either mixed or non-competitive inhibition. We conclude that the MAO-B inhibitory activity in urine cannot be attributed to isatin alone and another inhibitor(s) appears to be present.