EPHB2, an ephrin receptor (EPH) from receptor tyrosine kinase (RTK) family, is one of the substrates for tissue factor (TF) - coagulation factor VIIa (FVIIa) complex and it is cleaved in its ectodomain. EPHB2 cleavage is important for ephrin receptor (EPH) - ephrin ligand (EFN) signaling and cell repulsion. TF has been reported to be overexpressed in different cancer types such as breast and colorectal cancer (CRC). Furthermore, EPHB2 R155C mutation, at the TF/FVIIa-mediated cleavage site, has been identified as one of the somatic mutation sites in human metastatic CRC. Therefore, the aim of the present work was to characterize the EPHB2 R155C mutation and its effect on the cleavage by TF/FVIIa on EPHB2 in context to CRC. We generated overexpression cell models for EPHB2 wild type (wt) and R155C mutant in human CRC DLD-1 cell line for in vitro compartmentalization assay analysis to demonstrate repulsion event in EPH-EFN signaling. Whereas low endogenous TF expression led to incomplete cleavage of EPHB2 wt protein, stable overexpression of TF resulted in complete cleavage. Moreover, overexpression of TF resulted in reduced compartmentalization in EPHB2 wt cells after FVIIa treatment. Transient expression of TF in EPHB2 wt and R155C cells showed no clear difference in EPHB2 cleavage. Interestingly, it was difficult to obtain similar stable overexpression level of TF in EPHB2 R155C cells compared to EPHB2 wt cells. This may lead to further research in context to the role of TF/FVIIa-mediated EPHB2 cleavage in CRC by the generation of TF overexpression cell lines using lentiviral transduction.