Reconstituted fibrillary collagen is one of the most advantageous biomaterials for biomedical applications. The objective of the research project described in this paper was to evaluate whether riboflavin-induced photo-crosslinking could be used as a non-toxic alternative to glutaraldehyde (GA)-crosslinking for the preparation of wet spun collagen filaments. Collagen filaments were produced on a laboratory wet spinning line and crosslinked with GA or riboflavin with and without UV exposure. Based on mechanical and thermal analyses, it was concluded that the combination of riboflavin and UV light leads to crosslinked collagen filaments having improved mechanical and thermal properties. Furthermore, riboflavin-crosslinked filaments exhibited a higher cytocompatibility for human mesenchymal stem cells compared to glutaraldehyde crosslinked filaments.