The endothelium is a key player for the function of every blood-perfused tissue and organ. Far from forming an inert barrier, endothelial cells play critical role in homeostasis and diseased states, including: blood flow regulation, vessel formation, action as a secretome, circulating blood cell adhesion amongst others. Dependent on their residing organ, endothelial cells exhibit a structural, phenotypic, and functional heterogeneity. With the advent of novel techniques able to interrogate the transcriptome, epigenome, and proteome at a single-cell resolution, cellular heterogeneity has been uncovered within tissue. These technical advances in profiling have also propelled our understanding of endothelial biology, with novel insights into their role in development, homeostasis, and disease. This Collection will focus on the study of endothelial cells at a single-cell level across species: human, murine, and others. The scope will cover both sequencing- and imaging-based techniques. Examples include but not limited to single cell (sc) scRNA-seq, scATAC-seq, single molecule fluorescent in situ hybridization (smFISH), and spatial transcriptomics and proteomics. The protocols will describe initial sample preparation, subsequent processing, and dataset analysis with a special emphasis on endothelial cells. Our goal through this is to provide standardized protocols for researchers interested in endothelial cell mapping and profiling.