Gelatin-based hydrogels have gained significant attention due to their tunable physicochemical properties and biocompatibility, making them ideal for biomedical and industrial applications. However, their gelation ability and mechanical properties vary depending on the amino acid content of gelatin, its concentration, and external conditions such as temperature. This study integrated experimental and molecular dynamics (MD) simulation approaches to understand the gelation behavior of crosslinker-free gelatin as a function of concentration and temperature. Characterization of the prepared gelatin solutions was done using dynamic light scattering, Fourier-transform infrared spectroscopy, mass spectrometry (MS), and swelling ratio tests. MD simulations were conducted at various concentrations and temperatures using gelatin sequence; one obtained from a published study and two identified through MS/MS analysis of porcine skin gelatin. The results showed that while temperature and concentration influenced the conformational properties, these changes were not significant. As the temperature rose, hydrogen-bonding between gelatin chains increased, whereas the number of hydrogen-bond formed with the water molecules decreased. However, high concentrations enhanced both types of hydrogen bond formation. At low temperatures (gel), more hydrogen bonds form compared to high temperatures (sol), highlighting gelatin’s sensitivity to temperature and its stronger interaction with water during gel formation. Furthermore, increasing the concentration improved the solvent’s surface exposure, favoring gelatin-water interactions. Swelling ratios were greater at lower concentrations under gelation conditions, aligning with experimental results. The integration of experimental and computational methods has offered important insights into gelation, along with identifying the conditions including amino acid sequences needed to effectively prepare crosslinker-free gelatin-based hydrogels.