Lipid droplets (LDs) are the main storage organelles for triglycerides. Elucidation of lipid accumulation mechanisms and metabolism are essential to understand obesity and associated diseases. Adipogenesis has been well studied in murine 3T3-L1 and human Simpson-Golabi-Behmel syndrome (SGBS) preadipocyte cell lines. However, most techniques for measuring LD accumulation are either not quantitative or can be destructive to samples. Here, we describe a novel, label-free LD quantification technique (LipiD-QuanT) to monitor lipid dynamics based on automated image analysis of phase contrast microscopy images acquired during in vitro human adipogenesis. We have applied LipiD-QuanT to measure LD accumulation during differentiation of SGBS cells. We demonstrate that LipiD-QuanT is a robust, nondestructive, time- and cost-effective method compared with other triglyceride accumulation assays based on enzymatic digest or lipophilic staining. Further, we applied LipiD-QuanT to measure the effect of four potential pro- or antiobesogenic substances: DHA, rosiglitazone, elevated levels of D-glucose, and zinc oxide nanoparticles. Our results revealed that 2 µmol/l rosiglitazone treatment during adipogenesis reduced lipid production and caused a negative shift in LD diameter size distribution, but the other treatments showed no effect under the conditions used here.