By allowing for direct injection of urine, liquid chromatography-tandem mass spectrometry (LC-MS-MS) with atmospheric-pressure chemical ionization has proven to be useful for analysis of drugs of abuse in human urine. The purpose of this study was to evaluate this technique by direct screening for 23 different substances (phenylethylamines, hypnotics, and N-benzylpiperazine) in urine samples. It was possible to achieve lower detection limits compared with commercial immunochemical methods. There was a linear response for all analytes with an intraday coefficient of variation of about 16%, and the gradient elution gave a variability in relative retention time of about 1%. Positive results were confirmed by reanalysis including sample preparation by solid-phase extraction. Among the 529 authentic urine samples analyzed, 35 samples were screened positive for phenylethylamines, and 20 for hypnotics. Of these, 23 (66%) samples were confirmed to be positive for phenylethylamines, and 11 (55%) for hypnotics. This study demonstrates that LC-MS-MS is a valuable complement to immunochemical screening analysis, especially for substances for which immunochemical methods are not yet available or when an increased sensitivity is needed.