Sensitive imaging techniques for small animals are needed to assess drug toxicity in preclinical studies. Optical coherence tomography (OCT) provides a noninvasive tool for high-resolution, depth-resolved visualization of drug-induced changes in tissue morphology. In a mouse model, we utilize OCT to assess vaginal tissue integrity following the application of topical microbicides (drugs used to prevent infection). Mice are challenged with herpes simplex virus-2 (HSV-2) to determine the correlation of tissue damage as quantified by OCT to increased susceptibility. The microbicide benzalkonium chloride (BZK) (0.02, 0.2, or 2%) or phosphate buffered saline control is administered intravaginally. In vivo OCT imaging and collection of tissue samples are performed after treatment. A quantitative OCT scoring system is applied to assess epithelial damage, and the results are compared with those of histology. A separate group of mice are treated similarly then challenged with HSV-2. Epithelial morphology quantified noninvasively by OCT and histology are dose-dependent (p<0.0001). The OCT scoring system detected a significant increase in epithelial damage with increasing BZK concentration (p<0.0001). These results paralleled an increase in HSV-2 susceptibility (p<0.005). OCT can be used as a noninvasive tool to assess topical drug toxicity in a small animal model with potential to predict increased susceptibility to vaginal infection.