Human monocytes can be derived from the leukocyte-rich by-product of donors' blood available after platelet separation. Large volumes of the monocyte samples obtained from this product provided an opportunity to conduct experiments with relatively high concentrations of the antimicrobial agents sufficient for their detection in bioassays, thus avoiding the necessity of working with the radiolabelled drugs. Washing of the cells after their exposure to the drug may lead to an extraction of the tested agent from the cell, especially if it is a substance of low molecular weight. In our experiments we excluded the washing step, and separated the monocytes from the extracellular medium by velocity gradient centrifugation. In experiments with two rifamycins, the cell pellet as well as the extracellular fluid were subjected to a bioassay using Micrococcus luteus as a target organism. The method showed good reproducibility and consistency in results obtained.