Background: Vulvar squamous cell carcinoma (VSCC) is treated with wide local excision. The challenge is to remove as much skin as necessary to prevent recurrence, but meanwhile preserve genital skin to diminish morbidity. Optical coherence tomography (OCT) is a noninvasive imaging tool that produces cross-sectional images. Optical coherence tomography could be helpful in determining appropriate surgical margins during excision of VSCC. Objective: This study aimed to assess the value of OCT in determining appropriate surgical margins in patients operated for VSCC. We hypothesize that benign tissue will differ qualitatively (presence of clear epidermal layers) and quantitatively (epidermal layer thickness and attenuation coefficient) from (pre)malignant tissue. Materials and Methods: In 18 patients with a pretreatment biopsy of VSCC, before excision, areas within the center (tumor), at the margin (skin next to the center), and in normal vulvar skin outside the area of resection were imaged by OCT. Optical coherence tomography data were assessed on the presence of a clear epidermal layer, thickness of the epidermal layer, and values of µOCT. Results were grouped according to histopathological report in a benign group and a (pre)malignant group. Results: A clear epidermal layer was observed in all OCT images of benign tissue and only in 6 of 23 premalignant lesions (P < 0.001). The epidermal layer thickness as well as the µOCT was significantly smaller for benign vulvar tissue than for (pre)malignant tissue (0.29 vs 1.03 mm, and 2.4 vs 4.1 mm-1, respectively; P < 0.001). The diagnostic accuracy of OCT, as calculated by receiver operating characteristic curve analysis, showed at defined thresholds a sensitivity of 100% and specificity of 80% when considering layer thickness, and a sensitivity of 100% and specificity of 70% when considering the attenuation coefficient. Conclusions: We show that qualitative and quantitative OCT imaging can distinguish between benign and (pre)malignant vulvar tissue, enabling appropriate surgical margin detection with noninvasive in vivo OCT imaging.