We developed an analytical method based on solid phase extraction, on-support derivatization and isotope dilution-GC/MS for the detection of dialkyl phosphate (DAP) metabolites, dimethyl thiophosphate, diethyl thiophosphate, dimethyl dithiophosphate, and diethyl dithiophosphate in human urine. The sample preparative procedure is simple and fully automated. In this method, the analytes were extracted from the urinary matrix onto a styrene-divinyl benzene polymer-based solid phase extraction cartridge and derivatized on-column with pentafluorobenzyl bromide. The ester conjugated analytes are eluted from the column with acetonitrile, concentrated and analyzed. Compared to extraction-post extraction derivatization methods for the analysis of DAP metabolites, this on-support derivatization is fast, efficient, and less labor-intensive. Furthermore, it has fewer steps in the sample preparation, uses less solvent and produces less interference. The method is highly sensitive with limits of detection for the analytes ranging from 0.1 to 0.3 ng/mL. The recoveries were high and comparable with those of our previous method. Relative standard deviation, indicative of the repeatability and precision of the method, was 1-17% for the metabolites.