Rhizoma Alismatis (RA) was wildly used for treatment of dysuria, pyelonephritis, hyperlipidemia, enteritis diarrhea, diabetes, inflammation, and cancer. Triterpenoids are the major active components of RA, and its extract is mainly composed of alisol A (ALA), alisol B (ALB), alisol C 23-acetate (ALC-23A), alisol A 24-acetate (ALA-24A), and alisol B 23-acetate (ALB-23A). In this study, a simple, reliable, and sensitive ultra high-performance liquid chromatography with triple quadrupole mass spectrometry (UHPLC–MS/MS) method was created and validated for the quantification of the five major triterpenoids in rat plasma and various tissues biosamples (including intestine, stomach, liver, kidney, fat, muscle, brain, heart, lung, spleen, and testes). The plasma and tissues biosamples were pretreated by direct precipitation deproteinization method with acetonitrile. 17α-Hydroxyprogesterone was used as internal standard (IS). The chromatography was performed on a Phenomenex C8 column (30 × 2.00 mm, 1.8 μm) at room temperature with gradient elution. Compounds were quantified by selected multi-reactions monitoring (SRM) scanning with positive electric spray ionization mode. The linearity of detection for each triterpene was respectively from 1 to 1000 ng/mL for ALC-23A and ALA, from 4 to 4000 ng/mL for ALA-24A, from 10 to 10,000 ng/mL for ALB, and from 2 to 2000 ng/mL for ALB-23B (r > 0.99) with low quantification limits of 1–10 ng/mL for all analytes. All of the other validation parameters were also in an acceptable range. The validated UHPLC–MS/MS method subsequently applied for the pharmacokinetic and tissue distribution studies of RA extract. After orally given 100 mg/kg of RA extract, ALA was the most exposed component, followed by ALB and ALA-24A. Whereas significant gender difference was observed for ALB, ALA, and ALA-24A between female and male rats. The AUC(0-∞) of ALA, ALB, and ALA-24A in female rats were approximately 2–5 fold larger than that in male rats. These triterpenoids also displayed approximately 1.5–2 times longer half-life (t1/2) in female rats. Appearant Km, Vmax and Clint of ALA, ALB, and ALA-24A were calculated by substrate depletion approach, rat P450 CYP3A2 plays an important role in the metabolism of ALA, ALB, and ALA-24A, which is an important factor leading to the different exprosures of ALA, ALB, and ALA-24A between the male rats and the female rats. Furthermore, results from tissue distribution in male rats showed that the main tissue depots of five triterpenoids were the stomach/intestine, followed by the liver, brain, and fat. However, ALA was still measured in the kidney after a long elimination time. ALB and ALB-23B exhibited lower elimination rate in the testis. These results provide a fundamental support for further pharmacological development and clinical safety application of RA.