Assessment of cell adhesion and cell size provides valuable information on surface biocompatibility. However, most investigations on cell morphology dynamics are time and resource consuming, of rather descriptive character and lack procedures for appropriate quantification. The aim of the study was to develop a software programme which allows automated cell segmentation and identification as well as calculation and further processing of cell size in low-contrast images. The software utilises modified edge detection and morphologic operations for automatic cell analysis in light microscopy images. In an application study, osteogenic cell-adhesion dynamics were quantified for the ECM proteins collagen type I (COL) and fibronectin (FIB) over a period of 12 hrs. Untreated tissue culture polystyrene (TCPS) served as control. The software programme proofed full function in automatic cell tracking and quantification of cell size. After 11 h, cell sizes were highest for COL (6391 ± 1167 µm(2)) and FIB (6036 ± 411 µm(2)) compared with TCPS (3261 ± 693 µm(2)). The developed software allows quantification of initial cell size changes on translucent surface modifications and is suitable as a reliable tool for fast biocompatibility screening. Osteogenic cell adhesion was significantly promoted by COL and FIB indicating the potential of respective functionalized biomaterial surfaces.