Vulvar squamous cell carcinoma (VSCC) is a gynecological cancer with an incidence of two to three per 100,000 women. VSCC arises from vulvar intraepithelial neoplasia (VIN), which is diagnosed through painful punch biopsy. In this study, optical coherence tomography (OCT) is used to differentiate between normal and VIN tissue. We hypothesize that (a) epidermal layer thickness measured in OCT images is different in normal tissue and VIN, and (b) quantitative analysis of the attenuation coefficient () extracted from OCT data differentiates VIN from normal vulvar tissue. Twenty lesions from 16 patients are imaged with OCT. Directly after data acquisition, a biopsy is performed. Epidermal thickness is measured and values of are extracted from 200 OCT scans of normal and VIN tissue. For both methods, statistical analysis is performed using Paired Mann–Whitney-test. Correlation between the two methods is tested using a Spearman-correlation test. Both epidermal layer thickness as well as the are different between normal vulvar tissue and VIN lesions (). Moreover, no correlation is found between the epidermal layer thickness and . This study demonstrates that both the epidermal thickness and the attenuation coefficient of vulvar epithelial tissue containing VIN are different from that of normal vulvar tissue.