Over the last two decades proteins have become increasingly important in human therapy and diagnosis. Engineering therapeutic proteins through improving their biological activity and stability has been a major interest in our group. In this mini-review we summarize our research on three proteins with pharmaceutical potential - serine protease inhibitor from squash seeds (CMTI), bovine pancreatic trypsin inhibitor (BPTI), and human fibroblast growth factor 1 (FGF1). To improve the functional properties of these proteins we used multiple techniques such as homology approach, rational design, total chemical synthesis, site-directed mutagenesis and phage display. The physicochemical properties of the obtained protein variants were evaluated using protein crystallography, spectroscopic techniques, enzymatic assays, stability measurements as well as numerous biological tests.