Liquid chromatography plays the important and critical role in the field of clinical pharmacology of evaluating drugs in biological matrices. Studying antiretroviral drugs in the female genital tract has important implications for using drugs as a vaginal microbicide for prevention of HIV-1 sexual transmission. Accurate measurement of drug levels is extremely important in optimizing drug concentration in gel formulation. Extracting drugs from small volumes of viscous, proteinaceous substances like cervicovaginal fluid (CVF) is a practically challenging process. The proposed method was designed to introduce procedure for sample collection and drug extraction procedure for CVF matrix before the chromatographic separation. Based on this extraction method, we validated a reverse phase high performance liquid chromatography with electrospray ionization mass spectrometry assay in order to quantify UC781 in female genital tract compartment. The LC-MS method was validated based on a novel extraction technique which proved to be efficient in reducing analyte degradation with an average extraction efficiency of 72%. This method is accurate, demonstrating an average accuracy over three QC (n = 30) concentrations ranging from 99.9% to 106.1%. Average precision within-day and between-day ranged from 3.1% to 10.2% and 5.1% to 6.4%, respectively. We demonstrated that the analyte was able to maintain its stability under various conditions using this extraction method. The sample preparation, extraction, and the powerful liquid chromatography and mass spectrometry can readily be applied for accurate quantification of similar drugs in CVF.