Abstract Aim: An LC-MS/MS method to quantify drug in dried cervicovaginal secretions from flocked swab was developed and validated using the antiretroviral efavirenz as example. Methods: Cervicovaginal swabs (CVS) were prepared by submerging flocked swabs in efavirenz-spiked matrix. Time to full saturation, weight uniformity, recovery and room temperature stability were evaluated. Chromatographic separation was on a reverse-phase C18 column by gradient elution using 1mM ammonium acetate in water/acetonitrile at 400 µL/min. Detection and quantification were on a TSQ Quantum Access triple quadrupole mass spectrometer operated in negative ionisation mode. The method was used to quantify efavirenz in CVS samples from HIV-positive women in the VADICT study (NCT03284645). Results: Swabs were fully saturated within 15 seconds, absorbing 128 µL of matrix with coefficient of variation (%CV) below 1.3%. The method was linear with a weighting factor (1/X) in the range of 25-10000 ng/mL with inter- and intra-day precision (% CV) of 7.69-14.9%, and accuracy (% bias) of 99.1-105.3%. Mean recovery of efavirenz from CVS was 83.8% (%CV, 11.2) with no significant matrix effect. Efavirenz remained stable in swabs for at least 35 days after drying and storage at room temperature. Median (range) CVS efavirenz AUC0-24h was 16370 ng*h/mL (5803-22088), Cmax was 1618 ng/mL (610-2438) at a Tmax of 8.0 h (8.0-12), and Cmin was 399 ng/mL (110-981). Efavirenz CVS:plasma AUC0-24 ratio was 0.41 (0.20-0.59). Conclusion: Further application of this method will improve our understanding of the pharmacology of other therapeutics in the female genital tract, including in low- and middle-income countries.