Polysaccharide lyases are a diverse group of enzymes that degrade uronic acid-containing polysaccharides via a β-elimination mechanism. They are produced by a variety of organisms belonging to various domains of life such as bacteria, fungi, and marine animals. Polysaccharide lyases cleave a variety of substrates derived from bacteria, plants, and animals. They have immense applications in the food processing industry, textile industry, and therapeutics. This chapter catalogs various enzymes belonging to the family of polysaccharide lyases and their major sources, production, purification, characterization, and assay methods. The current demand in industry and therapeutics is for robust and efficient polysaccharide lyases. This can be achieved by protein engineering, strain improvement, and immobilizing the enzymes. This chapter also focuses on various strain-improvement methodologies and concludes with the future aspects of polysaccharide lyases.