Gelatin-based hydrogels are widely used in biomedical fields because of their abundance of bioactive motifs that support cell adhesion and matrix remodeling. Although inherently bioactive, unmodified gelatin exhibits temperature-dependent rheology and solubilizes at body temperature, making it unstable for three-dimensional (3D) cell culture. Therefore, the addition of chemically reactive motifs is required to render gelatin-based hydrogels with highly controllable cross-linking kinetics and tunable mechanical properties that are critical for 3D cell culture. This article provides a series of methods toward establishing orthogonally cross-linked gelatin-based hydrogels for dynamic 3D cell culture. In particular, we prepared dually functionalized gelatin macromers amenable for sequential, orthogonal covalent cross-linking. Central to this material platform is the synthesis of norbornenefunctionalized gelatin (GelNB), which forms covalently cross-linked hydrogels via orthogonal thiol-norbornene click cross-linking. Using GelNB as the starting material, we further detail the methods for synthesizing gelatin macromers susceptible to hydroxyphenylacetic acid (HPA) dimerization (i.e., GelNB-HPA) and hydrazone bonding (i.e., GelNB-CH) for on-demand matrix stiffening. Finally, we outline the protocol for synthesizing a gelatin macromer capable of adjusting hydrogel stress relaxation via boronate ester bonding (i.e., GelNB-BA). The combination of these orthogonal chemistries affords a wide range of gelatin-based hydrogels as biomimetic matrices in tissue engineering and regenerative medicine applications.