A reliable liquid chromatography/tandem mass spectrometry has been developed for simultaneous evaluation of the activities of five cytochrome P450s (CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A) in rat plasma and urine. The five-specific probe substrates/metabolites include phenacetin/paracetamol (CYP1A2), tolbutamide/4-hydroxytolbutamide and carboxytolbutamide (CYP2C9), mephenytoin/4′-hydroxymephenytoin (CYP2C19), dextromethorphan/dextrorphan (CYP2D6), and midazolam/1′-hydroxymidazolam (CYP3A). Internal standards were brodimoprim (for phenacetin, paracetamol, midazolam and 1′-hydroxymidazolam), ofloxacin (for 4′-hydroxymephenytoin, dextromethorphan and dextrorphan) and meloxicam (for tolbutamide, 4-hydroxytolbutamide and carboxytolbutamide). Sample preparation was conducted with solid-phase extraction using Oasis^® HLB cartridges. The chromatography was performed using a C₁₈ column with mobile phase consisting of methanol/0.1% formic acid in 20 mM ammonium formate (75:25). The triple–quadrupole mass spectrometric detection was operated in both positive mode (for phenacetin, paracetamol, midazolam, 1′-hydroxymidazolam, brodimoprim, 4′-hydroxymephenytoin, dextromethorphan, dextrorphan and ofloxacin) and negative mode (for tolbutamide, 4-hydroxytolbutamide, carboxytolbutamide and meloxicam). Multiple reaction monitoring mode was used for data acquisition. Calibration ranges in plasma were 2.5–2500 ng/mL for phenacetin, 2.5–2500 ng/mL for paracetamol, 5–500 ng/mL for midazolam, and 0.5–500 ng/mL for 1′-hydroxymidazolam. In urine calibration ranges were 5–1000 ng/mL for dextromethorphan, 0.05–10 μg/mL for dextrorphan and 4′-hydroxymephenytoin, 5–2000 ng/mL for tolbutamide, 0.05–20 μg/mL for 4-hydroxytolbutamide and 0.025–10 μg/mL for carboxytolbutamide. The intra- and inter-day precision were 4.3–12.4% and 1.5–14.8%, respectively for all of the above analytes. The intra- and inter-day accuracy ranged from −9.1 to 8.3% and −10 to 9.2%, respectively for all of the above analytes. The lower limits of quantification were 2.5 ng/mL for phenacetin and paracetamol, 5 ng/mL for midazolam, 0.5 ng/mL for 1′-hydroxymidazolam, 5 ng/mL for dextromethorphan, 50 ng/mL for dextrorphan and 4′-hydroxymephenytoin, 5 ng/mL for tolbutamide, 50 ng/mL for 4-hydroxytolbutamide and 25 ng/mL for carboxytolbutamide. All the analytes were evaluated for short-term (24 h, room temperature), long-term (3 months, −20 °C), three freeze–thaw cycles and autosampler (24 h, 4 °C) stability. The stability of urine samples was also prepared with and without β-glucuronidase incubation (37 °C) and measured comparatively. No significant loss of the analytes was observed at any of the investigated conditions. The current method provides a robust and reliable analytical tool for the above five-probe drug cocktail, and has been successfully verified with known CYP inducers.