PURPOSE: To evaluate the cytotoxicity of reusable cannulas for ophthalmic surgery after the cannulas were filled with an ophthalmic viscosurgical device (OVD) and cleaned with an enzymatic detergent. SETTING: Microbiological Testing Laboratory, Department of Medical-Surgical Nursing, University of São Paulo School of Nursing, and Cell Culture Section, Adolfo Lutz Institute, São Paulo, Brazil. DESIGN: Experimental study. METHODS: The sample consisted of 30 reusable 25-gauge injection cannulas, 20.0 mm in length, whose lumens were filled with an OVD solution for 50 minutes. The following steps were used to process the cannulas: (1) presoaking, (2) washing the lumen using a high-pressure water jet, (3) backwashing with enzymatic detergent in ultrasonic cleaner, (4) preliminary rinsing with tap water, (5) final rinsing with sterile distilled water, (6) drying with compressed filtered air, (7) wrapping in surgical-grade paper, and (8) steam sterilization at 134°C for 4 minutes. The cannulas were then tested for cytotoxicity according to the United States Pharmacopeia 32. RESULTS: The cleaning protocol used in this study removed residues of OVD solution and enzymatic detergent as shown by the lack of cytotoxicity of all sample extracts. CONCLUSION: This cleaning protocol has the potential to minimize the occurrence of toxic anterior segment syndrome associated with residues of OVD solutions and enzymatic detergents. FINANCIAL DISCLOSURE: No author has a financial or proprietary interest in any material or method mentioned.