Affibody molecules are small and robust non-immunoglobulin affinity ligands capable of binding to a wide range of protein targets. They are selected from combinatorial libraries based on a 58 amino acid, three-alpha-helical Z-domain scaffold. They share no sequence or structural homologies to antibodies and in contrast to antibodies they can be functionally produced both by peptide synthesis and by recombinant expression in Escherichia coli. Protein engineering is used to adapt Affibody molecules binding to a target of interest to the specific demands imposed by the intended application. Obviously, the optimal molecule for molecular imaging will be different from the optimal molecule for therapy. Here, we describe general strategies to optimize Affibody molecules for diagnostic imaging and therapy applications.