Background: Piperaceae family are a well-known source of structurally diverse amides with the wide range of bioactivities such as cytotoxic, stomach aches, insect repellents, anti-inflammatory, insecticidal, and antifeedant activities. It has been reported that piplartine and piperine, alkaloid/amide compounds from Piper species, show antitumor activities. Objective: A rapid, sensitive liquid chromatography-tandem mass spectrometry method has been developed and validated for the determination of piplartine and piperine from Piper chaba extract. Materials and Methods: The two analytes, together with internal standard (IS, trichostachine), were separated on a Waters Acquity ethylene bridged hybrid C18(2.1 mm × 100 mm, 1.9 μ) column using a mobile phase of acetonitrile with 0.1% formic acid and water with 0.1% formic acid (70:30, v/v) with isocratic elution. The detection was performed using the positive ion electrospray ionization in multiple reaction monitoring mode with transitions at m/z 318→221 for piplartine, m/z 286→201 for piperine, and m/z 272→201 for the IS. Results: The calibration curves were both linear (r2 > 0.995) over a concentration range of 1.0–2000 ng/mL; the lower limit of detection quantification was 1.0 ng/mL for both piplartine and piperine. The intra-day and inter-day precisions (relative standard deviation %) were <10.9%, and recoveries ranged from 90.3% to 103.0%. Conclusions: The analytes were proven stable in the short-term, long-term, and after three freeze-thaw cycles. The method was successfully applied to pharmacokinetic studies of piplartine and piperine in rats after oral administration of P. chaba extract.