Although determination of circulating endothelial progenitor cell (EPC) in peripheral blood by flow cytometry is an emerging marker for cardiovascular medicine, a common standardized protocol is still not available, due to the low numbers achieved in peripheral blood. In the present paper we describe a novel technique for EPC quantification as CD34+/CD144+/CD3- cells within the lymphocyte gate, which increases the percentages of EPC positivity described before and also offers high intra-assay reproducibility. These improvements are based on a gating strategy for big-sized lymphocytes, smooth fixation and cytometric clearance of CD3+ lymphocytes (T-cells). This last procedure is able to increase intra-assay Pearson's correlation from 0.8517 to 0.8908. Therefore, the technical setting described here offers a high-performance and clinically oriented EPC determination strategy in human peripheral blood.