This protocol regards a microscopic application and software for image-guided monitoring of mammalian cells which grow in suspension cultures. It has been developed in order to establish an automated microscopic application for in situ and at-line cell monitoring in bioreactors (Akin et al., Biosens Bioelectron 26:4532-4537, 2011; Babitzky et al., At-line microscopic analysis of suspension cell cultures. In: ECCE/ECAB, the first joint European Congress of chemical engineering and applied biotechnology, September 25-29, 2011, Berlin, Germany, 2011. http://www.tci.uni-hannover.de, Poster). The application aims to assess the analysis of an appropriated sample volume of mammalian cell cultivation medium. The sample is injected into a microfluidic slide which is suitable for transmitted light microscopy and is attached to an automated microscope device, the at-line microscope. The major attribute of microscope automation ascribes to the camera software, which enables sequential image capturing and storing. Image analysis and cell detection are performed by the software that is based on the edge detection algorithm developed by Canny (IEEE Trans Pattern Anal Mach Intell 8:679-698, 1986; Finding edges and lines in images.Technical Report 720, MIT Artificial Intelligence Laboratory, 1983). The analysis results are cell count, morphological characteristics, and grayscale values of the detected cells. The presented setup can be applied to low-volume cultivations and has been successfully tested for monitoring CHO-K1 cell cultivation processes.