A rapid screening method was developed for the determination of phenothiazines by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF–MS). In this method, α-cyano-4-hydroxy cinnamic acid (CHCA) was used as the matrix to assist the ionization of phenothiazines. The identification of 11 phenothiazines with heavy side chains was achieved by observing their protonated molecular ions [M+H]+ at m/z 340–447. Quantification was achieved by using triflupromazine at m/z 353 as the internal standard (IS). The relative ionization efficiencies of 11 phenothiazines and IS on MALDI-TOF–MS were different from those achieved by ESI-MS, but the product ion spectra from MALDI-MS–MS were quite similar to those from ESI-MS–MS except in the case of flupentixol. The limit of detection was 0.3 ng/ml with a quantification range of 1–50 ng/ml urine in the best case; the limit of detection was 5 ng/ml with a quantification range of 10–100 ng/ml urine in the worst case for 10 phenothiazines except thiethylperazine. The present method provides a simple and high-throughput method for the screening of these phenothiazines using only 20 μl of urine. To our knowledge, this study is the first trial to analyze phenothiazines by MALDI-TOF–MS (–MS).