Ocriplasmin, a truncated form of plasmin, is commercialized in the USA and in Europe under the trade name Jetrea®, and indicated for the treatment of symptomatic vitreomacular adhesion and vitreomacular traction including when associated with macular hole ≤400 μm, respectively. We have shown in a previous study that ocriplasmin undergoes autolytic degradation when injected in eye vitreous, which leads to its rapid inactivation. In order to investigate this process further, we have introduced in ocriplasmin a variety of amino acid substitutions within or in the immediate vicinity of the three major autolytic cleavage sites. We demonstrate here that autolytic inactivation of ocriplasmin is a sequential process where initial cleavage occurs primarily between residues 156 and 157. Reduction or even blocking of autolysis can be achieved by mutating a limited number of key residues. In this study, we also report the identification of a series of ocriplasmin variants with improved resistance to autolysis and unimpaired catalytic activity. Such variants represent useful tools for the exploration of therapeutic approaches aiming at non-surgical resolution of vitreomacular adhesion.