Gelatin methacrylate (GelMA) is a widely used biomaterial in tissue engineering and regenerative medicine. GelMA is a chemically modified form of gelatin. Researchers have employed various methods to synthesize GelMA, such as the conventional method (Bulcke et al. 2000), the sequential method (Lee et al. 2015), and facile one-pot (Shirahama et al. 2016) methods to achieve GelMA hydrogels with a wide range of degree of functionalization or methacrylation. However, the impact of these different synthesis methods and their reaction parameters on GelMA hydrogels and scaffolds remains to be investigated concerning bioink formulation and 3D printing application. In this study, we prepared GelMA hydrogels using different synthesis protocols and used several analytical techniques to compare the physical, chemical, and rheological properties of the differently synthesized GelMA hydrogels and their biological responses. We also formulated a GelMA/gelatin-based hydrogel for 3D printing and characterized printing parameters for all the different sets of hydrogels. The outcome of this comparative study showcases that while GelMA synthesized with the conventional method can be used for 3D modeling of both soft and hard tissue, GelMA synthesized with the sequential and facile one-pot method would be best suited for modeling soft tissue and hard tissue, respectively, owing to their hydrogel property and post photocrosslinking stability. Therefore, depending on the tissue engineering application of the GelMA-based scaffolds, researchers can refine their GelMA synthesis protocol and 3D printing approach for GelMA-based bioinks. © 2022