Purpose: Challenges exist regarding antiretroviral quantitation in the female genital tract. Endocervical wicking using sterile tear flow test strips is an alternative to conventional methods due to the consistent sample volume obtained. Methods: A novel method for measuring antiretrovirals in cervicovaginal secretions using Sno-strip® wicking was developed and tested by spiking Sno-strips® with known concentrations of tenofovir, nevirapine, atazanavir, lopinavir, and ritonavir in blank cervicovaginal lavage fluid. Drug concentrations were determined by high-performance liquid chromatography with ultraviolet or mass spectrometry detection. Results: Mean extraction recoveries were 91% for tenofovir, 89% for nevirapine, 63% for atazanavir, 60% for lopinavir, and 61% for ritonavir relative to controls. Freezing spiked samples for 24 hours at -80°C had no effect on recovery. Conclusions: Results suggest that the antiretrovirals tested can be efficiently extracted from Sno-strips®, although a greater percentage of tenofovir and nevirapine was recovered. Storage of Sno-strip® samples up to 24 hours before analysis showed no difference in the percentage of drug recovered compared with immediate analysis. Quantitating antiretroviral penetration into the female genital tract may assist in determining optimal therapeutic antiretroviral regimens to both decrease the risk of HIV transmission and prevent development of HIV drug resistance. © 2009 Thomas Land Publishers, Inc.