n this study, a high-performance liquid chromatographic method (HPLC) was developed, validated and applied for the determination of raltegravir in biological sample like saliva. Liquid- liquid extraction was performed for isolation of the drug and elimination of saliva interferences. Samples of saliva was extracted with 50µL of ortho phosphoric acid and 3ml of methanol was added and spiked with raltegravir. The chromatographic separation was performed on Agilent Eclipse C18 (100 mm × 4.6 mm, 3.5µm) column, by using 80:20 v/v acetonitrile: water as a mobile phase under isocratic conditions at a flow rate of 1.0 mL/min for UV detection at 240 nm. Retention time of raltegravir was found to be 1.030 min. Linearity was found to be in the range of 25-1000 ng/mL with regression equation y = 13864x + 40495 and correlation coefficient 0.999. The low % RSD value indicates the method is accurate and precise. The limit of detection (LOD) and limit of quantification (LOQ) were found to be 0.76 and 2.28 ng/mL, respectively. It can be concluded that this validated HPLC method is easy, precise, accurate, sensitive and selective for determination of raltegravir in saliva.