Directed evolution of proteins for improved or modified functionality is an important branch of modern biotechnology. It has traditionally been performed using various in vitro methods, but more recently, methods of in vivo artificial evolution come into play. In this review, we discuss and compare prokaryotic and eukaryotic-based systems of directed protein evolution in vivo, highlighting their benefits and current limitations and focusing on the biotechnological potential of vertebrate immune cells for the generation of protein diversity by means of the immunoglobulin diversification machinery.