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The present study was carried out to evaluate the in-vitro biocompatibility of acellular pericardium and diaphragm of caprine and bovine (buffalo) origin, bovine posterior aorta and porcine urinary bladder. They were made acellualr using different protocols and were cross-linked with different cross-linking agents. The crosslinking agents included glutaraldehyde (GA), glyoxal (GO) Diphenyl phosphoryl azide (DPPA), Ethylene glycol diglycidyl ether (EGDGE), polyethylene glycol (PEG), 1-ethyl-3(3-dimethyl aminopropyl) carbbodiimide (EDC) and hexamethylene diiisocyanate (HMDC) and 1,4-butanediol diglycidylether (BDDGE) for 12,24,48 and 72h. In-vitro studies included the gross observations, non-enzymatic degradation, enzymatic degradation, free protein determination, free amino group determination, fixation index, moisture percent analysis and SDS-PAGE analysis. The results of each biomaterials crosslinked with different agents were presented and discussed on the basis of in-vitro biocompatibilty determination.