Background: Osteoarthritis (OA) is a chronic protracted inflammatory joint disorder that frequently coexists with other comorbidities. Previous studies analyzed peptidase activity in synovial fluid in knee OA (KOA) patients, but they did not investigate whether type of therapy influences enzyme activity. Moreover, it is yet unknown if the activity of any particular enzyme can be taken into account as a reliable prognostic marker for the severity and progression of KOA. Objective: The aim of the current study is to assess synovial fluid peptidase's potential role as a biomarker for KOA in Egyptian patients as well as to determine whether it can predict the clinical progression of such disease. Patients and methods: A cross-sectional study was estimated on 75 KOA patients (Ahlback grade 3 or higher and indicated for arthrocentesis), who were subjected to sociodemographic and clinical characteristics, as well as Functional and Physical Health Status assessed every month. Puromycin sensitive aminopeptidase (PSA), neutral aminopeptidase (NAP), prolyl endopeptidase (PEP), aminopeptidase B (APB) glutamyl aminopeptidase (GLU), aspartate aminopeptidase (ASP), and pyro glutamyl aminopeptidase (PGAP) are among the peptidases shortlisted for their activity as biomarkers in synovial fluid. Results: A significant association between NAP (P=0.038) and APB (P=0.05) peptidases and pain generation on movement in patients with KOA was noticed. Only ASP peptidase showed significant correlation with range of motion (P=0.049). Another clinical condition of knee failure significantly influenced by the presence of ABP (P=0.013) and PEP (P=0.033) peptidase presence. When diagnosing knee effusion, PEP (P=0.050) and PGAP (P=0.018) differ significantly depending on the clinical symptom. PSA was protective (P=0.003), whereas GLU and PEP were risk factors, according to a binary logistic regression (P values 0.001 and 0.019, respectively). Conclusion: Peptidase activity is considered an important synovial fluid biomarker to determine disease progression in osteoarthritic patients, which could aid in early disease detection and the development of better treatment protocols.