To measure the concentration of brilliant blue G (BBG) in vitreous and plasma after use as a surgical adjuvant for staining and peeling of the internal limiting membrane to determine potential systemic adverse effects. This study was designed as a prospective, interventional, clinical, case series. Five eyes from five patients with macular hole or epiretinal membrane underwent BBG-assisted internal limiting membrane and epiretinal membrane removal. The vitreous samples were obtained and stored at the end of surgery in all five cases. The plasma specimens were extracted and stored at the end of the operation, after 4 hours, and after 7 days post operation. For BBG analysis of plasma and vitreous, high-performance liquid chromatography coupled with tandem mass spectrometric detection was used. Brilliant blue G was not detected in plasma from all five cases at the three points of measurement. The mean vitreous BBG concentration was 34.5 ± 23.7 ng/mL (range, 11.3-70.9 ng/mL). Postoperative progress was good, and adverse effects were not observed in any of the five cases. Brilliant blue G, which remained at low levels in the vitreous cavity, was not found in the systemic blood flow after the operation. Thus, any adverse effects of systemic BBG would be avoided.