A reversed-phase high performance liquid chromatographic method (HPLC) was established for determination of isothiocyanates in human urine, which used the reaction of propyl isothiocyanate with 1,2-benzenedithiol as reference instead of the synthesis of 1,3-benzodithiole-2-thione. The linear regression of the standard curve was determined to be y = 0.418 2 x + 2.821 (r2 = 0.999 3), that was well matched with the regression of methyl isothiocyanate, y = 0.412 2x + 2.442 3(r2 = 0.996 6). The detection limit (at approximately S/N = 2.5) was 0.08 micromol/L. The mean relative standard deviation (RSD) (n = 21) for the intra-day reproducibility was (4.7 +/- 2.9)%. The mean RSD (n = 4) for inter-day reproducibility of three samples was (7.0 +/- 5.0)%. The recovery of four propyl isothiocyanate standards was (91.3 +/- 7.9)% (mean RSD 8.7%). The initial results obtained by this method may provide useful information regarding to the kinetics of isothiocyanates in dietary consumption and excretion of individuals.