A simple and rapid quantitative UPLC–MS/MS method for simultaneous determination of picfeltarraenins IA and IB in rat plasma was developed and validated in accordance with the US FDA Bioanalytical Guidance (2001). Analytes were extracted from rat plasma by using methanol and separated on Agilent ZORBAX SB-C18 (50 mm × 2.1 mm, 1.8 μm) column by using a mobile phase composed of methanol and water (70:30, v/v). Eluents were monitored by ESI tandem mass spectrometry detection with SRM mode using ion transitions m/z 785.4 → 639.5, m/z 815.5 → 669.5, and m/z 763.5 → 455.3 for picfeltarraenin IA, picfeltarraenin IB, and internal standard, respectively. The method was validated over the linear range of 11.5–1150 ng/mL and 13.0–1300 ng/mL. The developed analytical method was applied to support a pharmacokinetic study on simultaneous estimation of picfeltarraenins IA and IB in rats.