Localizing, identifying and quantifying lipid droplets are important features to be assessed in the research of mechanisms involved in diseases such as steatosis, obesity, diabetes, myopathies and arteriosclerosis. Lipid-droplet staining with Oil Red O is widely used combined to either light field, conventional fluorescence microscopy and phase contrast microscopy. Here, for the first time, we report an easy, fast and precise protocol for the quantitative evaluation of lipid droplets staining with Oil Red O in HepG2 cells in vitro using confocal laser scanning microscopy associated with maximum intensity projection technique and counting point method. Our methodology was compared to a previous described protocol to measure lipid droplets, based on phase contrast microscopy and binarization. Our protocol substantially enhanced the quality of lipid droplets images compared to phase contrast microscopy by different reasons, such as: 1-Use of maximum intensity projection technique; 2- Out-of-focus light absence; 3-Increased contrast; 4-Enhanced lipid droplet definition. Thus, the use of this protocol increases the sensitivity of lipid droplets quantification, showing morphological results that can be underestimated using other approaches.