Abstract: Methods for simultaneously detecting multiple drug classes in a biological sample are described herein. In one embodiment, the biological sample is taken from an animal, such as a human. Suitable classes of drugs include drugs prone to abuse and prescription medications. In one embodiment, the biological sample is enzymatically hydrolyzed to liberate free drug in the sample. Once the biological sample has been prepared, the sample is extracted using solid-phase extraction (SPE). Following solid phase extraction (SPE), the resulting eluate containing the compounds to be detected is diluted and injected into a liquid chromatograph coupled to a triple quadrupole mass spectrometer. Multiple classes of drugs can be analyzed simultaneously in less than about 13 minutes, preferably less than about 11 minutes, more preferably less than about 10 minutes, more preferably less than about 9 minutes, most preferably less than about 8 minutes.