Layer-by-Layer technology (LbL), a technique based on the deposition of oppositely charged polyelectrolytes layer-wise on the surface of interest, has gained increasing interest in the field of drug delivery. The presented work in this thesis explores new applications for Layer-by-Layer (LbL) technology in the field of controlled protein delivery. The basic motives for these studies were the protein-friendly mild formulation conditions where only aqueous solutions are used, in contrast to organic solvents typically employed in the fabrication of many other protein formulations, thus correct protein folding and activity are preserved. Several formulations were designed for the loading and slow release of model and therapeutic biologicals from different carriers. Special attention was paid to testing the activity of released fractions. Moreover, double chambered nanoparticles were developed to host and co-deliver a protein and a small molecule drug to the same cells.