A sensitive and specific assay method for the simultaneous quantitation of 17α-hydroxyprogesterone caproate (17-OHPC), 17α-hydroxyprogesterone (17-OHP), and progesterone (P) in human plasma using high-performance liquid chromatography and tandem mass spectrometry (LC–MS/MS) was developed and validated. Plasma samples were processed by a solid phase extraction (SPE) procedure using Oasis® HLB extraction cartridge prior to chromatography. Medroxyprogestrone acetate (MPA) was used as the internal standard. The compounds were separated using Waters C18 Symmetry analytical column (3.5 μm, 2.1 mm × 50 mm) using a gradient elusion with a mobile phase consisting of 5% methanol in water [A] and methanol [B], with ammonium acetate (2 mM) and formic acid (0.1%) being added to both [A] and [B], at a flow rate 0.3 ml/min. The retention times for 17-OHPC, 17-OHP, P and MPA were 4.5, 1.5, 2.5 and 2.2 min, respectively, with a total run time of 7 min. The analytes were detected by a Micromass Quattro Micro triple quadrupole mass spectrometer in positive electron spray ionization (ESI) mode using multiple reaction monitoring (MRM). The extracted ions monitored following MRM transitions were m/z 429.10 → 313.10 for 17-OHPC, m/z 331.17 → 97.00 for 17-OHP, m/z 315.15 → 109.00 for P and m/z 387.15 → 327.25 for MPA (IS). The assay was linear over the range 1–200 ng/ml for 17-OHPC and 17-OHP, and 2–400 ng/ml for P, when 0.4 ml of plasma was used in the extraction. The overall intra- and inter-day assay variation was <15%. No significant variation in the concentration of 17-OHPC, 17-OHP or P was observed with different sample processing and/or storage conditions. This method is simple, allows easy, accurate and reproducible measurement of 17-OHPC, 17-OHP and P simultaneously in human plasma, and is used to evaluate the pharmacokinetics of 17-OHPC in pregnant subjects.