The emergence of medicinal indications for stem cell therapies has seen a need to develop the manufacturing capacity for adherent cells such as mesenchymal stem cells (MSCs). One such development is in the use of microcarriers, which facilitate enhanced cell densities for adherent stem cell cultures when compared with 2D culture platforms. Given the variety of stem cell expansion systems commercially available, novel methods of non-invasive and automated monitoring of cell number, confluence, and aggregation, within disparate environments, will become imperative to process control, ensuring reliable and consistent performance. The in situ epi-illumination of mouse embryonic fibroblasts and human mesenchymal stem cells attached to Cytodex 1 and 3 microcarriers was achieved using a bespoke microscope. Robust image processing techniques were developed to provide quantitative measurements of confluence, aggregate recognition, and cell number, without the need for fluorescent labeling or cell detachment. Large datasets of cells counted on individual microcarriers were statistically analyzed and compared with NucleoCounter measurements, with an average difference of less than 7% observed from days 0 to 6 of a 12-day culture noted, prior to the onset of aggregation. The developed image acquisition system and post-processing methodologies were successfully applied to dynamically moving colonized microcarriers. The proposed system offers a novel method of cell identification at the individual level, to consistently and accurately assess viable cell number, confluence, and cell distribution, while also minimizing the variability inherent in the current invasive means by which cells adhered to microcarriers are analyzed. (c) 2017 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.