Heme peptides and their derivatives, also called microperoxidases (MPs), are employed as heme protein active site models, catalysts, and charge-transfer chromophores. In this work, two approaches to the biosynthesis of novel MPs are described. In one method, heme peptides are expressed as C-terminal tags to the protein azurin and the MP is liberated by proteolytic cleavage by an endopeptidase. In an alternative approach, heme peptides are expressed as N-terminal tags to the cysteine protease domain (CPD) of the Vibrio cholerae MARTX toxin. Once activated by inositol hexakisphosphate, CPD undergoes autocleavage at an N-terminal leucine residue to liberate the MP. Purification is aided by use of a histidine-immobilized Sepharose column that binds exposed heme [Asher, W. A., and Bren, K. L. (2010) Protein Sci. 19, 1830-1839]. These methods provide efficient and adaptable routes to the preparation of a wide range of biosynthetic heme peptides.