Cancer cell behaviour such as motility, migration and adhesion are important parameters to study, in order to understand the properties of metastasis. The sialylation of proteins and lipids on cell surfaces is central for movement of cells. We have investigated two different cell lines with and without sialylation, MDAMB-231 and PC-3 Wild type (WT) and Simple Cells (SC) and used digital holographic cytometry (DHC) to monitor the morphological changes of these cells in vitro. DHC is a convenient method to monitor different cell types and to collect morphological data such as area and thickness. MDA-MB-231 and PC-3WT cells differed morphologically as shown by DHC. In terms of proliferation, after incubation for 30 h a significant difference was observed between the genetically engineered breast MDA-MB-231 SC line and the MDA-MB-231 WT cell line, while no significant differences in proliferation patterns were observed between prostate PC-3 WT and SC. The cellular area could be compared with data generated by conventional flow cytometry. Differences between the analyzed cell lines are also demonstrated by feature diagrams. Wound healing assays were conducted, and the MDA-MB-231 WT cell line significantly outperformed the PC-3 WT cell line during 24 h of incubation. For MDA-MB-231 WT/SC and the PC-3 WT/SC lines, respectively, no significant difference was observed in this assay. Furthermore, a motility analysis using DHC indicateda significant difference between MDA-MB-231 and PC-3 cells. A significant difference over time could be demonstrated between MDA-MB-231 SC and WTcells, as MDA-MB-231 WT cells accumulated further compared to MDA-MB-231 SC. A significant difference was also observed between PC-3 WT and PC-3SC, where the PC-3 SC motility increased over time.