Background/Aim: CD44 and CD133 have been implicated as biomarkers of cancer cells and their expression could be analyzed to identify circulating tumor cells. Although CD44 and CD133 have been shown to be expressed in prostate cancer cells, a differential expression pattern has been reported depending on the tumor stage and cell line examined. We further investigated CD44 and CD133 expression in different prostate cancer cell lines to confirm whether their expression is distinguishable among patients with various tumor stages. Materials and Methods: CWR22Rv1, PC3, LNCaP, and DU145 cell lines were cultured and the cell morphology was observed for three days. The single expression of CD44 or CD133 and their combined expression were analyzed by flow cytometry. Results: We report that the single expression of CD133 was less than 5% in all cell lines examined here. PC3 and DU145 cells displayed a high expression of CD44 (>93%), while the expression of CD44 was less than 4% in CWR22Rv1 and LNCaP cells. CWR22Rv1 was the only cell line that demonstrated a high co-expression of both CD44 and CD133. Conclusion: Both single and combined expression of CD44 and CD133 should be considered when validating the detection of prostate cancer cells in circulating tumor cells.