Purpose To demonstrate the role of vitrectomy-assisted biopsy with immunohistochemistry in the diagnosis and treatment of intraocular masses. Methods Academic video. Results Primary and metastatic intraocular tumors from the anterior and posterior segment are included. The role of incisional and excisional biopsy was achieved by partial lamellar sclerouvectomy, endoresection, vitreous sampling and resection of peripapillary tumor, allowing the diagnosis in uveal melanoma of iris and ciliary body choroid; likewise, diffuse retinoblastoma, juxtapapillary hemangioblastoma and choroidal metastasis from the genitourinary system and lung. A three-port 23- or 25-gauge system allowed the biopsy to be assisted by the vitrectomy cutter and a syringe connected to the aspiration line. The procedure led the treatment by excision, laser thermotherapy and cryotherapy. Conclusions Vitrectomy-assisted biopsy with immunohistochemistry is a useful tool in diagnosing intraocular masses, leading to the identification of primary tumor and serving as a gateway for local treatment.