Analyses of standard iminodipeptides and iminodipeptides in the urine of patients with prolidase deficiency have been demonstrated using liquid chromatography-mass spectrometry with an atmospheric pressure ionization interface system. The separation was carried out on a reversed-phase column using 0.1% aqueous trifluoroacetic acid-methanol (70:30 or 80:20). Very intense quasi-molecular ions [( M + H]+) of various standard iminodipeptides were observed by this method. The quasi-molecular ions [M + H]+ of various iminodipeptides were also observed in the urine samples of patients with prolidase deficiency, and Gly-Pro, Ala-Pro, Val-Pro, Leu-Pro, Ile-Pro, Ser-Pro, Thr-Pro, Glu-Pro, Asp-Pro, His-Pro, Lys-Pro, Pro-Pro and Tyr-Pro as iminodipeptides containing proline with C-terminal residue and Glu-Hyp, Pro-Hyp, Ile-Hyp and Gly-Hyp as iminodipeptides containing hydroxyproline with C-terminal residue were identified in the urine of patients with prolidase deficiency.