There are at least two distinct types of adipose cells and tissue in mammals, white and brown. White adipocytes store fat as triacylglycerol during caloric excess and release free fatty acid during fasting. By contrast, brown adipocytes are highly adapted to expend energy as heat via the action of Uncoupling protein-1 (UCP1) in the mitochondria. Brown adipocytes burn substantial amounts of glucose and lipid and can thus counteract obesity, diabetes and dyslipidemia. Significant amounts of brown adipose tissue have been recently identified in adult humans raising the possibility that variations in brown fat function may profoundly influence body weight and systemic metabolism. In addition, a subpopulation of progenitor cells is present within white adipose tissue that is capable of differentiating into brown like adipocytes or “beige/brite” adipocytes. We aim to establish a set of phenotypic analysis for identifying and developing small or large molecules that enhance differentiation of human adipose stem cells (hASCs) into “beige/brite” cells. Molecule(s) with such properties could potentially be beneficial for body weight as well as glycemic control for diabetic patients. A large scale isolation procedure has been established to obtain and bank hASCs from human adipose tissue biopsies in order to provide sufficient number of cells for screening. We focused on optimization of image-based medium thought-put assays using hASCs and UCP-1/neutral lipid as readout to screen and characterize new chemical entities (NCE’s) that encourage the “beige/brite” phenotype.These cells could be differentiated in 96-384 well plates into adipocytes with different UCP-1 content, which is detected by immunohistochemsitry using UCP-1-specific antibodies. This allows us to utilize high content imaging technology to quantitatively measure UCP-1 content and other morphological features. We will present results of using this technology to assess a set of reference compounds that potentially regulate the “browning” of adipose-derived stem cells. Positive hits from such screen will then been further characterized using Seahorse Extracellular Flux (XF) Analyzer in order to understand the effects of compound(s) on the bioenergetics. Patient consent: Samples of adipose tissue is collected from patients undergoing elective surgery at Sahlgrenska University Hospital in Gothenburg, Sweden. All study subjects received written and oral information before giving written informed consent for the use of the tissue. The studies were approved by The Regional Ethical Review Board in Gothenburg, Sweden