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In-vitro biocompatibility determinatuion of acellular dermal matrix cross-linked with 0.6 % Gluatraldehyde (GA), 1% Hexamethylene diiisocyanate (HMD), 1% 1,4-butanediol diglycidyl ether (BDE) and 1% 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) for 6, 12, 24, and 48h duration at room temperature was carried out in this study. The uncross-linked dermal matrix was used as control. The dermal matrix was made acellular using ionic biological detergent and then crosslinked. In-vitro studies included enzymatic degradation (collagenase, elastase and trypsin), free amino group contents, moisture contents, SDS-PAGE analysis, free protein contents and in-vitro immunocompatibility determination.The results showed that the EDC-48 crosslinked dermal matrix performed better as compared to other crosslinked matrices. The sequence of the rate of enzymatic degradation was GA<EDC<BDE<HMD<Native skin. More or less this sequence was observed in all the other parameters.