In this report, effects of reaction parameters on kinetic resolution of racemic atenolol using Pseudomonas fluorescens lipase were investigated via transesterification for production of pharmacologically active eutomer (S)-atenolol with high enantiomeric purity. It was found that a temperature of 45 °C produced an acceptable enantioselectivity (E: 17). Good agitation speeds were found at 170–230 rpm producing E values of 12–15, whilst an enzyme activity of ≥2500 U gave 100% conversion of the (S)-atenolol. Substrate concentrations of 11.26–18.80 mM gave E values of 11.6–12.3. Variation of the substrate molar ratio yielded (S)-atenolol conversions of 44.67–61.58% with E = 12–23.