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In this study in-vitro evaluation of cellular and acellular caprine pericardium following crosslinking with glutaraldehyde (GA), glyoxal (GO) diphenyl phosphoryl azide (DPPA), ethylene glycol diglycidyl ether (EGDGE) was carried out. The uncross-linked native and acellular pericardium was used as control. Fresh pericardium was divided into to equal halves. One part was cross-linked as such while another part was made acellular and then cross-linked. In-vitro studies included the gross observations, non-enzymatic degradation, enzymatic degradation (1% cyanogen bromide, pepsin and papain), free protein determination, free amino group determination, fixation index, moisture percent and SDS-PAGE analysis. Present study revealed that 0.65% GA and 1% GO, DPPA and EGDGE with a duration of 24 h at room temperature showed better cross-linking as compared to other combinations. GO treatment showed highest resistance to non-enzymatic and enzymatic degradation at different time interval. Significant reduction (P<0.05) of free amino group was observed with GA and DPPA treatment at different time interval as compared to control. Moisture percentage after GA treatment showed significant reduction (P<0.01) at different time intervals when it was compared to control. GO treatment resulted in better cross-linking and formation of high molecular weight protein as evaluated by SDS-PAGE.