The determination of mercapturic acids, as metabolic end-products of an important pathway in the biotransformation of xenobiotics, in human urine is becoming a standard tool for the biomonitoring of populations exposed to potential mutagens and carcinogens. Mass spectrometric methods permit the direct identification and characterization of these substances without a previous derivatization step. In this work, N-acetylcysteine conjugates of synthetic origin were characterized by three different mass spectrometric methods: matrix-assisted laser desorption/ionization (MALDI) mass spectrometry, positive and negative californium-252 plasma desorption mass spectrometry (PD-MS) and fast-atom bombardment (FAB) mass spectrometry. For PD-MS, samples were prepared by the spin deposition technique on plain aluminium targets. α-Cyano-4-hydroxycinnamic acid and 2,5-dihydroxybenzoic acid were used as MALDI matrices and glycerol as a matrix for FAB. Complementary structural information on the N-acetylcysteine moiety and the xenobiotic moiety was obtained through PD-MS and FAB spectra. All three techniques could be used for relative molecular mass determination. The spectra exhibited protonated molecules [M + H] + or [M + Na] + ions with high abundance. MALDI was the most sensitive among the three tested methods.