Biocatalysis has emerged in the last decades as a valuable alternative to the use of classical catalysts for the synthesis of chiral valuable compounds. The use of free enzymes or whole cells as biological catalysts in chemical processes presents a wide set of advantages, as excellent selectivity can be achieved while working under mild and environmentally friendly conditions. Different approaches can be performed while applying biocatalysts in asymmetric synthesis, including the kinetic resolution of a racemate, the application of dynamic processes as the dynamic kinetic resolution, the use of parallel kinetic resolution, the deracemization of starting materials, or the desymmetrization of prochiral substrates. In the present chapter, the recent advances of these techniques while using biocatalysts for the synthesis of high-added value molecules have been reviewed, also analyzing the application of multicatalytic systems, well combining several biocatalysts or one biocatalyst with other types of catalysts, for the preparation of chiral compounds avoiding work-up, isolation, and purification steps.