With the development of the two step desolvation method for the preparation of gelatin nanoparticles, it is possible to reproducibly generate homogeneous colloidal spheres. After the establishment of a surface modification to achieve stable nucleotide loading onto the particles, the goal of this study was to test this new biodegradable and simple producible non-viral gene delivery system in vitro. Two different types of gelatin nanoparticles, referring to size and zeta (ζ) potential were investigated. Therefore, we varied conditions as for example the loaded DNA amount and the conjugation media to find a preferable setup. All preparations were tested on B16F10 murine melanoma cells using pCMVLuc as reporter gene. To evaluate our results, we used commonly known, but non-biodegradable, polyethylenimine (PEI) polyplexes as "gold standard" for in vitro transfection. Additionally, we performed accompanying cell viability assays and hemolysis studies with the preparations tested to substantiate the thesis of low cell toxicity of gelatin nanoparticles. Different setups resulted in efficient gene delivery. The achieved levels of gene expression were good but lower as with optimized PEI polyplexes. Nevertheless, the already achieved results show gelatin nanoparticles as promising biodegradable alternative to existing non-viral gene delivery systems.