BACKGROUND AND OBJECTIVE: Classic vitreous biopsy, which targets the vitreous with an undiluted sample of 1 mL to 2 mL, has been used as a diagnostic analysis. Vitrectomy under air infusions have been reported to be able to extract more vitreous sample. In this study, the authors introduce a way of obtaining vitreous sample under air irrigation using 23-gauge vitrectomy and discuss the benefits and potential risks of this procedure. PATIENTS AND METHODS: In this retrospective case series study, a total of 65 eyes of 65 patients with macular epiretinal membrane (ERM) or macular hole (MH) were enrolled. A vitreous biopsy was carried out with air infusion. Vitrectomy with fluid infusion was then carried out to remove the residual vitreous. Medical records of patients with macular ERM or MH were reviewed and analyzed. Clinical data, including age, sex, best-corrected visual acuity (BCVA), optical coherence tomography (OCT), axial length, presence of posterior vitreous detachment (PVD), presence of liquefication of vitreous, and refraction, were recorded and investigated. The volume of vitreous sample, visual outcome, and complications related to vitreous biopsy at 1-month follow-up were recorded and analyzed. RESULTS: The mean of undiluted vitreous sample volume was 2.1 mL +/- 0.2 mL. There were seven patients whose vitreous samples were less than 2 mL during the vitreous biopsy. The mean age of patients was 62.9 years +/- 8.4 years (range: 35 years to 85 years) at diagnosis. There were 18 male and 47 female patients. At 1-month follow-up, no patient had decreased visual acuity. There was one patient who had a peripheral retinal break and was treated with photocoagulation during the operation (1.5%). The insufficient vitreous sample that may occur during the vitreous biopsy under air infusion was related to liquefication of vitreous (28.8%). CONCLUSION: In summary, vitreous biopsy with air infusion is a safe and effective maneuver to harvest undiluted vitreous in patients without significant vitreous inflammation.