Lipid droplets (LDs) are essential cytoplasmic organelles that allow both the storage and mobilisation of lipid, yet LD phenotypes (LD sizes/ numbers per cell) can vary greatly between cell types. As the deregulation of lipid metabolism is a hallmark of human cancers, we hypothesise that LD phenotypes in cancer cells could serve as biomarkers for sensitivity or resistance towards lipid metabolism-targeted therapies. In order to quantify LDs in a high-throughput setting, we measured BODIPY-stained LDs in human breast cancer (AU-565, HMC-1-8, EVSA-T, ZR-75-30, MDA-MB-231) and osteosarcoma cell lines (U2OS, 143B) using a MetaXpress High-Content Imaging system and analysis software. Under routine cell culture conditions, HMC-1-8 and AU-565 breast cancer cells showed the highest LD numbers/ cell (mean values of 43 LD/ cell and 15 LD/ cell, respectively) and the largest LD areas/ cell (mean values of 10.6 μm2 and 5.6 μm2, respectively). We then compared LDs in cells cultured with/ without 200 μM or 400 μM oleic acid supplementation for 24 hours, or with 0.5-2.0 μg/ml Brefeldin A (BFA) or DMSO vehicle for 5 or 24 hours. Oleic acid supplementation or BFA treatment for 24 hours significantly increased or reduced LD numbers/ cell, respectively, in all cell lines tested. However, after BFA treatment for 24 hours, we noted significant differences in cell survival between cell lines. These results indicate that high-throughput techniques can reproducibly quantify LDs in different cancer cell lines, and that measurements of LD phenotypes and cell survival could be combined for future large-scale drug screening.