Ammonia-lyases catalyze a wide range of processes leading to α,β-unsaturated compounds by elimination of ammonia. In this chapter, ammonia-lyases are reviewed with major emphasis on their synthetic applications in stereoselective preparation of unnatural amino acids. Besides the synthesis of various unnatural α-amino acids with the aid of phenylalanine ammonia-lyases (PALs) utilizing the 3,5-dihydro-5-methylidene-4H-imidazol-4-one (MIO) prosthetic groups, the biotransformations leading to various unnatural β-amino acids with phenylalanine 2,3-aminomutases using the same catalytic MIO prosthetic group are discussed. Cloning, production, purification, and biotransformation protocols for PAL are described in detail.