The endothelium plays a central role in cardiovascular regulation. Endothelial cells produce a variety of vasculoregulatory and vasculotropic molecules that act locally or at distant sites. Alteration of the vascular endothelium is a primary event in the pathogenesis of vascular diseases, such as atherosclerosis, as well as systemic and pulmonary hypertension. For these reasons, the study of human endothelium has become central in cardiovascular research. Unfortunately, technologies handling endothelial cells in vitro are often criticized due to the uncertain transferability of results to intact organs and, importantly, to humans. Although methods to assess endothelial function non-invasively have been available for decades, cell-based approaches for the direct ex vivo evaluation of endothelial cell biology in humans have been devised only recently. The discovery of endothelial progenitor cells opened the way for studies on vascular regeneration, while it has been recognized that mature circulating endothelial cells mainly represent a consequence of the ongoing vascular damage. Coupled with a minimally invasive way to obtain fresh human endothelial cells through an endovascular biopsy, these new methods provide a novel outlook on human endothelial cells as close as possible to their natural environment. In this review, we will summarize the current knowledge and the methodological perspective of these cell-based methods.