It has recently been proposed that plasma levels of 4β-hydroxycholesterol (4βHC) may be indicative of cytochrome P450 3A4 (P450 3A) activity and therefore could be used to probe for P450 3A-mediated drug-drug interactions. With this in mind, we describe a highly sensitive and precise liquid chromatography-electrospray ionization-tandem mass spectrometry method for the measurement of 4βHC in human plasma with a lower limit of quantification established at 2 ng/mL using 50 μL of plasma. The entire sample preparation scheme including saponification and derivatization of 4βHC to the corresponding dipicolinyl ester (DPE) was completed in less than 8 h using an automated sample preparation scheme enabling higher-throughput capabilities. Chromatographic resolution of 4βHC from 4α-hydroxycholesterol and other endogenous isobaric species was achieved in 11-min using an isocratic gradient on a C18 column. Because of endogenous concentrations of 4βHC in plasma, a stable isotope labeled (SIL) analogue, d7-4βHC, was used as a surrogate analyte and measured in the standard curve and quality control samples prepared in plasma. A second SIL analogue, d4-4βHC, was used as the internal standard. The intraday and interday accuracy for the assay was within 6% of nominal concentrations, and the precision for these measurements was less than 5% relative standard deviation. Rigorous stability assessments demonstrated adequate stability of endogenous 4βHC in plasma and the corresponding DPE derivative for the analysis of clinical study samples. The results from clinical samples following treatment with a potent P450 3A inducer (rifampin) or inhibitor (ketoconazole) are reported and demonstrate the potential future application for this highly precise and robust analytical assay.