Enantiomerically pure α-substituted β-hydroxyesters are important chiral building blocks for ligands, auxiliaries and β-lactam antibiotics. A two-step chemo-enzymatic procedure using lipase as biocatalyst is an efficient way to synthesize such products. To date, the methods described are limited to molecules that do not contain a chiral center adjacent to the racemic carbinol, and furthermore, they are limited to acylation. Here, we investigated the deracemization of diastereomerically pure syn- and anti-α-substituted β-hydroxyesters containing two stereo centers, using experimental methods under neat conditions and classical molecule dynamics (MD) simulation. A screening of free and immobilized commercial lipases identified immobilized lipase B from Candida antarctica (Novozyme 435) as the most appropriate biocatalyst for sterically demanding α-substituted β-hydroxyesters. Using Novozyme 435, reaction conditions were optimized and hydroxyesters (3S) or (3R) were achieved with enantiomeric excesses up to ≥99% ee and maximum overall yields of 80%. The absolute configuration of the enantiomers was eventually determined by ¹H-NMR spectroscopy after derivatization with MOSHER'S reagent (α-methoxy-α-trifluoromethylphenylacetic acid = MTPA).