In contrast to conventional silk fibroin, spider silk's potential as a scaffold material for tissue engineering is examined in this work. The remarkable qualities of spider silk are being researched for use in making films for tissue regeneration. In comparison to silk fibroin films, the study's analysis of orb-web spider Trichonephila clavata films highlights their improved cell adhesion and nanofibrous network structure. Tests for solubility substantiate the durability of spider silk films, while in vitro investigations demonstrate low cytotoxicity and enhance cellular viability. The conclusion highlights the exceptional properties of spider silk, which make it a viable option for tissue engineering applications and a step forward for in vitro cell culture and regenerative bioengineering.