Pharmacological vitrectomy refers to the use of enzymes in an effort to liquefy vitreous and to weaken the adhesion of vitreous cortex to the internal limiting membrane during or before performing vitreous surgery. It is well known that the vitreoretinal interface plays important roles in developing many blinding diseases. To make the vitreous surgery easier for better outcome or to avoid vitrectomy, plasmin, dispase, and chondroinase have been used to promote the disinsersion of vitreous cortex to the internal limiting membrane, a basement membrane of Muller cells. On the other hand, hyaluronidase has been used clinically to facilitate the clearance of vitreous hemorrhage liquefying vitreous body and developing posterior vitreous detachment. This article reviews enzymes as an intraoperative adjunctive agent in vitrectomy.