The purpose of this study was to improve the biocompatibility of glutaraldehyde (GA) cross-linked chitosan coated collagen scaffold for cartilage tissue regeneration. In order to prevent the potential toxicity of GA, we treated the designed scaffold with either glutamic acid or glycine. Amino acid treated scaffolds were characterized by scanning electron microscopy (SEM) techniques. Afterward, chondrocyte interaction with the composite scaffold was investigated assessing cell adhesion and proliferation using Hoechst staining and MTT cell proliferation assay, respectively. The SEM analyses of the scaffolds' surface and cross-section confirmed the adhesion of amino acids on the surface of the scaffolds. We also observed that scaffolds' porosity was reduced due to the coverage of the pores by chitosan and amino acids, leading to low porosity. The use of amino acid improved the chondrocyte adhesion and proliferation inside the scaffolds' pores when cells were cultured onto the chitosan-coated collagen scaffolds. Overall, our in vitro results suggest the use of amino acid to improve the biocompatibility of natural polymer composite scaffold being crosslinked with glutaraldehyde. Such scaffold has improved mechanical properties; biocompatibility thus may be useful for tissue regeneration such as cartilage.