We present a fabrication process to create bifunctional microparticles displaying two different proteins have been spatially segregated onto hemispheres. Silica and polystyrene microparticles with 2.0 m, 4.08 m, and 4.74 m diameters are processed with metal deposition to form two chemically distinct and segregated hemispheres. The surface of each hemisphere is then separately derivatized with proteins using different chemical conjugation strategies. These bifunctional Janus particles possess biologically relevant, native conformation proteins attached to a biologically-unreactive and safe substrate. They also display high densities of two types of spatially segregated proteins which may enable a range of capabilities that monofunctional particles cannot, such as improved targeting of drug carriers and bioimaging agents.