Lipid droplets (LDs) are dynamic organelles that regulate the storage and homeostasis of intracellular triglycerides and other neutral lipids. Studies show that the number, morphology, and subcellular localization of LDs are altered in a growing number of diseases. As such, methodologies for imaging and quantifying LDs have become essential tools for detecting changes in cellular lipid metabolism, which could be an important indicator of disease onset or progression. We previously reported on the accumulation of LDs in podocytes of the kidney glomerulus in nephrological diseases of metabolic and non-metabolic origin. Here, we describe a high-content analysis (HCA) method for automated detection and quantification of LDs in differentiated human podocytes.