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A simple high-performance liquid chromatography method for the determination of hydromorphone in small volume plasma has been developed. Following solid-phase extraction using Oasis HLB cartridges, samples were separated by reverse-phase high-performance liquid chromatography on an Atlantis T3 4.6 x 150 mm column (3.0 mu m) and quantified using mass spectrometry. The mobile phase was a mixture of water with 0.1% formic acid and acetonitrile with 0.1% formic acid (91: 9). The standard curve ranged from 1 to 500 ng/mL. Intra- and Inter-assay variability for hydromorphone was <10%, and the average recovery was >90%. The LLOQ was 1 ng/mL. This method was successfully applied to the analysis of hydromorphone samples at this institution. This method could be useful to those investigators dealing with small sample volumes, particularly when conducting pharmacokinetic studies that require multiple sampling from the same animal.