TOPIC: To assess the role and utility of intraoperative optical coherence tomography (iOCT) guidance during pars plana vitrectomy with chorioretinal biopsy for patients with suspicious chorioretinal lesions. CLINICAL RELEVANCE: Chorioretinal lesions suspicious for malignancy sometimes require surgical biopsy for diagnosis and treatment guidance. Biopsy site selection is currently guided by presurgical imaging, and clinical assessment. We sought to determine whether iOCT provides useful information for biopsy site selection to ensure a diagnostic specimen and assessment of wound repair after biopsy. MATERIALS AND METHODS: DISCOVER is a prospective study examining microscope-integrated iOCT systems in ophthalmic surgery. In this report, we examine all eyes in the DISCOVER study that underwent chorioretinal biopsy. Clinical characteristics, surgical technique, and imaging findings were reviewed. In addition, surgeon feedback data on iOCT utility and value to the procedure was also evaluated. RESULTS: Six eyes were identified within the DISCOVER study that underwent chorioretinal biopsy. iOCT was used to assess retinal and choroidal lesions prior to chorioretinal incision. Following biopsy, iOCT was utilized to assess retinal attachment and wound margin integrity. Additionally, iOCT was performed in real-time during biopsy to ensure proper biopsy depth and position. In 2 cases, preoperative OCT evaluation of retinal lesions was not possible. Utilizing iOCT, comprehensive evaluation of anatomic features was successfully performed prior to biopsy. This facilitated identification of biopsy sites most likely to provide a diagnostic sample based on lesion morphology on iOCT. After completion of chorioretinal biopsy, iOCT verified completeness of biopsy site and the retinal-RPE relationship (e.g., complete apposition, partial detachment). CONCLUSION: Microscope-integrated iOCT provides real-time guidance of biopsy site selection and verification of biopsy site stability following specimen collection.