Retinal detachment is a severe eye condition characterized by the detachment of the neurosensory retina from the retinal pigment epithelium and caused by retinal tears. Pars plana vitrectomy is the elective surgical procedure during which vitreous humor, a fluid which shapes the eye globe and supports mechanically the adherence of the retina to the posterior chamber of the eye, is collected. Therefore, profiling of the vitreous humor proteome of subjects diagnosed with retinal detachment is supposed to provide molecular evidence on the pathobiology of the disease and on its secondary complications, such as proliferative vitreo-retinopathy, which predispose to recurrent RD (observed in 20% of cases), a sight threatening condition. Herein, we investigated the perturbations of vitreous proteome between subjects affected by primary retinal detachment and controls by shot-gun proteomics approaches. Proteomic datasets were first analyzed and searched for global proteome changes. Thereafter, starting from the assumption that the disease could be sustained by altered proteolytic processing of structural and non-structural elements of the VH, mining of N- and C-termini was performed to uncover endogenous proteolytic events underscoring the disease condition. The search retrieved evidence of a wide repertoire of either previously characterized or uncharacterized proteolytic events. Most notably, comparison of the N- and C-termini landscape between experimental groups highlighted robust alterations in the repertoire of cleaved proteins. Further strengthened by immunoblotting studies on a select panel of proteins, the data envisage that retinal detachment is promoted or characterized, as a secondary process, by proteolytic-based alterations of structural and non-structural components involved in the regulation of immune process, proteases inhibition and most notably, angiogenesis.