Intracellular lipid droplets (LDs), subcellular organelles playing a role in long-term carbon storage, have immense potential in biofuel and dietary lipid production. Monitoring the state of LDs in living cells is of utmost importance for quick biomass harvest and screening promising isolates. Here, a deep-learning-based segmentation model was developed for automatic detection and segmentation of LDs using the model yeast species Lipomyces starkeyi, leading to fast and accurate quantification of lipid contents in liquid cultures. The trained model detected the yeast's cell and LDs in light microscopic images with an accuracy of 98% and 92%, respectively. Lipid content prediction using pixel numbers counted in segmented LDs showed high similarity to lipid quantification results obtained with gas chromatography-mass spectrometry. This automated quantification can highly reduce cost and time in real-time monitoring of lipid production, thereby providing an efficient tool in bio-fermentation.