Assessment of preactivated and thiolated silicone oils as potential long-term vitreous replacement. Thioglycolic acid (TGA) and 3-mercaptopropionic acid (MPA) were covalently coupled to amino-modified silicone oil and subsequently preactivated with 2-mercaptonicotinic acid (2-MNA). Each silicone thiomer was evaluated in view of oxidation, reductive reliquefaction, emulsification, and cytotoxicity. Both thiol ligands were coupled in almost quantitative yield to the silicone oils' backbone with a total amount of thiol groups of 223 ± 25 and 219 ± 11 µmol/g for silicone-TGA and silicone-MPA, respectively. A following preactivation with 2-MNA of around 50% of all thiol groups could be achieved. Preactivated silicones showed a protection against oxidation as the viscosity of silicone-TGA and silicone-MPA after iodine treatment were two- and fourfold higher than the preactivated counterparts, respectively. The percentage of emulsification was below 8% for both preactivated silicones in comparison to control with 100% emulsification. Silicone-TGA-MNA and silicone-MPA-MNA could be aspired via a 20 G needle within 35 s and showed a reversible sol-gel transition. Preactivated silicone oils tackle the shortcomings of currently available silicone oils as they unite suitable handling qualities with a resistance against emulsification, which renders them promising for long-term vitreous replacement. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2015. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 551-559, 2017.