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The asymmetric synthesis and kinetic resolution of a series of acyloins (α-hydroxy ketones) suitable as building blocks for the northern half of epothilones was studied. Three methods were applied to obtain nonracemic compounds at the eventual epothilone C15-position: asymmetric synthesis with Evans’ auxiliary, chemical resolution and enzymatic resolution. The success rate in small scale applications increased in the order given, and the enzymatic resolution was studied in more detail. Out of a set of nine lipases and esterases, lipases from Burkholderia cepacia, Pseudomonas sp., lipase B from Candida antarctica and recombinant esterases from Streptomyces diastatochromogenes exhibited the highest enantioselectivities with E-values ranging from 60 to >200. Pig liver esterase exhibited inverse enantiopreference and only with recombinant enzyme could a moderate selectivity (E = 50, commercial PLE: E = 8) be observed.