The analytical method permits simultaneous determination of various alkyl mercapturic acids in urine. For determination, the urine samples are buffered to pH = 2.5, spiked with isotope labeled internal standards (d3‐MeMA, d5‐EtMA, d7‐PrMA and d7‐iPrMA), processed using online solid phase extraction (online SPE) and analyzed. For that purpose the analytes are enriched on a C18 preconcentration column [Restricted Access Material (RAM) phase] and separated from most of the matrix. Subsequently, the analytes are transferred in backflush mode from the RAM phase to the analytical column, separated chromatographically and finally subjected to tandem mass spectrometric detection. For quantification, a calibration is carried out using standard solutions in pooled urine which are processed and analyzed in the same way as the samples to be analyzed.