To study the proteome of the subretinal fluid (SRF) from rhegmatogenous retinal detachment (RRD) in search for novel markers for improved diagnosis and prognosis of RRD.Human undiluted SRF obtained during vitrectomy for primary RRD using a 41-gauge needle (n = 24) was analyzed and compared to vitreous humor from 2-day postmortem eyes (n = 20). Sample preparation underwent nanoflow liquid chromatography-tandem mass spectrometry. Label-free quantification (LFQ) using MaxQuant was used to determine differentially expressed proteins between SRF and vitreous humor. The intensity-based absolute quantification (iBAQ) was used to rank proteins according to their molar fractions within groups. Identification of proteins beyond the quantitative level was performed using the Mascot search engine.The protein concentration of the control vitreous humor was lower and more consistent (1.2 ± 0.4 mg) than that of the SRF (17.9 ± 22 mg). The iBAQ analysis showed high resemblance between SRF and vitreous humor, except for crystallins solely identified in vitreous humor. The LFQ analysis found 38 protein misregulations between SRF and vitreous humor of which the blood coagulation pathway was found to be enriched using the PANTHER Classification System. Combined, the iBAQ, LFQ, and Mascot analysis found an overlap only in chitinase-3-like protein 1 and galectin-3-binding protein unique to the SRF.The proteome of the SRF was highly represented by proteins involved in proteolysis. Such proteins can possibly serve as targets in modulating the effects of SRF in RD.To identify potential novel biomarkers for therapeutic targeting in RD.