A novel bio-analytical method was developed and validated for the quantitative determination of rilpivirine in rat serum by using the liquid-liquid extraction chromatography and tandem mass spectrometric detection (HPLCMS/ MS). Separation of rilpivirine from the endogenous substances is achieved after liquid-liquid extraction by using HPLC-MS/MS system. Rilpivirine was eluted in isocratic mode with acetonitrile, methanol and 0.1% acetic acid in 5 mM ammonium acetate ( 20:25:55) at a flow-rate of 0.6 mL/min on Waters, Discovery C18, 50*4.6 mm, 5μm particle size column. Didanosine was used as the internal standard. The liquid-liquid extraction recovery was found 74% indicates good recovery. The validation results demonstrated that the present method was found to be precise and accurate. The stability tests indicated that the rilpivirine in rat serum is stable for three freeze-thaw cycles at both -20 ºC and -70 ºC, 18-h ambient storage, 15-day frozen storage at both -20 ºC and -70 ºC. The results also showed no significant matrix effect (<5.2%). The present method was found to be sensitive and selective at very low levels of linearity range 2-1000 ng/mL, based on a sample volume of 50 μL, with a linear correlation coefficient of ≥ 0.99. The validated method has been successfully applied to support a preclinical pharmacokinetic study.