The fragmentation of sixteen S-substituted N-acetyl(-l-)cysteine derivatives (mercapturic acids and methyl esters) was studied under collision induced dissociation (CID) conditions recording series of product (daughter) ion spectra. After transfer of the data from the MS data system to an external PC, a PASCAL computer program called PDNL (Parent — Daughter — Neutral Loss) was used a) to elucidate the structures of fragment ions by means of comparison with an MS/MS spectral library, b) to construct an MS/MS data domain, c) to eliminate consecutive decompositions, d) to evaluate the fragmentation pathways and e) to develop an MS/MS detector for mercapturic acids or their methyl esters. In this way common fragmentation reactions for mercapturic acids and their methyl esters could be ascertained. Selected reaction monitoring using a direct insertion probe was used to determine the concentration of 3-hydroxypropylmercapturic acid (compound 2) in human urine. The limit of detection was 50 ppb (signal-to-noise ratio=3:1) using 5 μl extracts from urine. This method was compared with a classical method for the quantitative determination of compound 2. The relative standard deviation and the coefficient of variation was specified for the MS/MS me