This study describes a highly sensitive, solid-phase extraction-HPLC/MS/MS assay for cyclophosphamide in human plasma, with a minimum quantifiable level of 0.025 μg mL−1. Plasma samples (0.5 mL) were mixed with 0.01 M ammonium acetate buffer (1.5 mL, pH 4.9) and internal standard (D4 deuterated compound, 50 μL, 5 μg mL−1), and then extracted using Isolute CH(EC) cartridges (100 mg). Contaminants were washed from the cartridges with 0.01 M ammonium acetate:methanol (90:10, 1 mL), prior to the elution of the compounds of interest with 0.1 M ammonium acetate:methanol (50:50, 300 μL). An aliquot (200 μL) of the eluent was injected onto the chromatography system, which consisted of a LiChroCART 4-4 RP-select B, 5 μm cartridge precolumn, protecting a LiChrospher 60 RP-select B, 5 μm (250 x 4 mm id) cartridge analytical column.