Human adipose tissue is the largest endocrine organ and plays a role in whole-body metabolism. Dysfunction of this tissue is involved in multiple diseases, such as obesity, diabetes, and cardiovascular disease. An important factor in maintaining healthy adipose tissue is ensuring correct functioning of the blood vessels in this highly vascularized tissue. The endothelial cells (ECs) which line blood vessels show remarkable heterogeneity in structure and function in physiological and pathological conditions. While multiple studies have been performed to characterize ECs in different organs, the endothelium of adipose tissue remains poorly characterized. One of the significant challenges in working with adipose tissue is the separation and isolation of single viable cells, including ECs. This chapter describes a reliable and flexible approach for the isolation of adipose ECs that could be used for various analysis, including single-cell RNA sequencing, in vitro culture, and downstream applications.