Enhanced fixation of bioprosthetic tissue by glutaraldehyde (GA) and additional cross-links with L-lysine significantly reduces calcification. Prolonged exposure to high-volume amino-compounds under warm, acidic conditions leads to thorough, non-rebounding GA detoxification. This study aimed to prove that removal of excess GA can amplify the benefits of enhanced GA cross-linking with regard to bioprosthetic tissue calcification. Porcine ascending aorta and leaflet tissue, and bovine pericardium, were fixed using 0.2%, 1.0%, 3.0% (v/v)) GA for seven days at 4° C. Samples were allocated to nine groups. Groups I to III received only GA treatment (one at each concentration); groups IV to IX underwent additional L-lysine treatment (48 h/37° C/0.1M) two days before completion of seven-day GA fixation; groups VII to IX underwent GA extraction using high-volume urazole solution followed by NaBH₄ reduction. Samples were implanted subcutaneously in rats for six weeks. Tissue calcium was measured by atomic absorption spectrophotometry and examined histologically after von Kossa staining. Calcification was reduced in all three tissue types by enhanced cross-linking and extraction of excess GA. Increasing GA concentrations from 0.2% to 3.0% led to reductions in calcification of 11.5% in leaflets, 63.6% in pericardium, and 17.5% in aortic wall tissue. Introducing additional cross-links with L-lysine led to significant reductions of calcium in all tissues (42.5% in leaflets, 79.3% in pericardium, 49.6% in aortic wall). Optimal reduction in calcification was achieved with combined 3.0% GA fixation/L-lysine/urazole detoxification. When compared with 0.2% GA-fixed tissue, calcification was reduced by 99.1% in leaflets, 95.9% in pericardium, and 90.8% in aortic wall tissue. In conclusion, removal of excess GA from fixed bioprosthetic tissue may markedly improve the anti-calcific effect of enhanced GA cross-linking.