Glutaraldehyde (GA) fixation of bioprosthetic tissue is a well adapted technique, with commercial products on the market for almost 40 years. Amine groups present in tissue react with GA to form different types of cross-links. An estimation of the degree of cross-linking of the tissue can be obtained by measuring the concentration of residual amine groups, which is frequently carried out with the 2,4,6 trinitrobenzene sulphonic acid (TNBS) assay. Cross-linked tissue and collagen matrices are usually further characterized by determining their physical properties (such as the shrinkage temperature), biological properties (such as resistance to enzymatic degradation), and mechanical properties before in vivo evaluation takes place. In an effort to improve the properties of cross-linked tissue and collagen, alternative cross-linking methods have been developed. One of these methods is based on the use of water soluble carbodiimides (CDI). It is generally accepted that this cross-linking method leads only to the formation of amide linkages between tissue carboxyl and amine groups. Therefore, until recently the TNBS assay was also used to determine the degree of cross-linking of CDI cross-linked tissue and collagen. However, it cannot be excluded that after activation of carboxyl groups of tissue and collagen by CDI, these groups can react with other nucleophiles (like hydroxyl groups) present in the matrix. To obtain a better insight in the degree of cross-linking of CDI cross-linked matrices a reliable assay for quantification of residual carboxyl groups is required. Up to now such an assay was not available. In this study a new assay to determine residual carboxyl groups in CDI cross-linked collagen matrices is presented. Reconstituted dermal bovine collagen matrices (RDBC) were cross-linked with a water soluble CDI and N-hydroxysuccinimide (NHS) and residual carboxyl groups were labeled using 5-bromomethyl fluorescein. Subsequently, the fluorescent label was released by mild hydrolysis and quantified with capillary zone electrophoresis. A calibration curve relating the concentration of carboxyl groups with peak intensities was obtained using SephadexTM standards with known concentrations of carboxyl groups. The concentration of carboxyl groups in unprocessed RDBC as determined with this new technique was equal to the concentration of carboxyl groups measured by amino acid analysis. On the basis of the concentration of residual carboxyl groups determined for CDI/NHS cross-linked RDBC and RDBC, in which the amine groups were blocked with propionaldehyde before CDI/NHS cross-linking, it was concluded that activated carboxyl groups can also react with other groups (such as hydroxyl groups) present in the matrix. This implies that the crosslink density of RDBC matrices after treatment with CDI/NHS is higher than expected on the basis of amide bond formation only, as determined by the TNBS assay.