Microtiter plates are commonly used for screening purposes and one-pot biotransformations. Nonetheless, there are scarce systematic studies focused on the application of these systems to complex whole cell multistep bioconversions, which typically require prolonged incubation periods, and on the evaluation of the reproducibility of data generated in such shaken miniature reactors. The present study aims to contribute to fill in this gap. The model system selected was the side-chain cleavage of β-sitosterol performed by whole cells of Mycobacterium sp. NRRL B-3805 in 24-well microtiter plates. Reproducibility of the data was assessed within the 24 wells of the microtiter plate as well as matching different plates placed in different positions on the shaking platform. Results show the suitability of microtiter plates with proper oxygen and pH monitoring capabilities to carry out complex multistep bioconversion using relatively slow growing bacteria. Reproducibility levels are in the range of an interval of confidence of 92%. © 2009 Elsevier B.V. All rights reserved.