The present study evaluates the crosslinking of electrospun gelatin nanofibers by physical and chemical methods to further elucidate the importance of the application of gelatin scaffold platforms for cell-based assays. The dehydrothermally cross-linked electrospun gelatin scaffolds were unable to retained their structure morphology and integrity upon exposure to 1X PBS or cell-culture media. The DHT and EDC/Sulfo-NHS cross-linked gelatin scaffolds exhibited fiber diameter on average in the nanometer range. Subsequently, we utilized 1X PBS and cell culture media to evaluate the stability of the nanofibers in solution. The immersion evaluation indicated that the chemically crosslinked gelatin nanofibers maintained their random nanofiber distribution and morphology. However, a high degree of swelling was observed in the presence of cell culture media. Overall, the gelatin scaffold demonstrated good performance in PBS and cell culture media. Hence, EDC/Sulfo-NHS crosslinked electrospun gelatin nanofibrous scaffolds have good biocompatibility and are promising bio-scaffolds for cell-based assays.