Efforts to develop biocatalysts with new chemical reactivity and selectivity different from that found in nature indicate that promising strategies involve incorporation of a metal-containing moiety into protein scaffolds. We have studied artificial metalloenzymes harboring a transition metal complex covalently-linked within a hydrophobic cavity of a rigid barrel protein, nitrobindin (NB), which perform C−C bond-forming reactions, photocatalytic hydrogen evolution, and others. Directed evolution of Cp*Rh(III)-linked NB, an artificial metalloenzyme, was achieved on the basis of an in vitro high-throughput screening (HTS) platform which involves an affinity purification step employing a starch-agarose resin for a maltose binding protein (MBP)-tag. Three rounds of directed evolution and screening of more than 4,000 clones in total yielded a Cp*Rh(III)-linked NB variant with an enhanced activity for cycloaddition of acetophenone oximes with alkynes. The HTS platform for directed evolution provides an efficient strategy for generating a highly active artificial metalloenzyme incorporating a synthetic metal cofactor. Along this line the artificial metalloenzyme with a protein scaffold fused with a helix-loop-helix domain on the basis of a covalent modification approach will be presented.