A barrier in scaling laboratory processes into automated microfluidic devices has been the transfer of laboratory based assays: Where engineering meets biological protocol. One basic requirement is to reliably and accurately know the distribution and number of biological cells being dispensed. In this study, a novel optical counting technique to efficiently quantify the number of cells flowing into a microtube is presented. REH, B-lymphoid precursor leukemia, are stained with a fluorescent dye and frames of moving cells are recorded using a charge coupled device ͑CCD͒ camera. The basic principle is to calculate the total fluorescence intensity of the image and to divide it by the average intensity of a single cell. This method allows counting the number of cells with an uncertainty Ϯ5%, which compares favorably to the standard biological methodology, based on the manual Trypan Blue assay, which is destructive to the cells and presents an uncertainty in the order of 20%. The use of a microdevice for vertical hydrodynamic focusing, which can reduce the background noise of out of focus cells by concentrating the cells in a thin layer, has further improved the technique. Computational fluid dynamics ͑CFD͒ simulation and confocal laser scanning microscopy images have shown an 82% reduction in the vertical displacement of the cells. For the flow rates imposed during this study, a throughput of 100-200 cells/s is achieved.