Lipid extraction from microalgae using a chloroform-methanol mixture was performed to obtain optimum conditions for the isolation of total lipids. The effects of extraction parameters including proportions of methanol to chloroform and the ratios of total solvent volume to dry biomass on extraction yields were evaluated. In addition, the effect of water on extraction performance was investigated for the purpose of applications to extract lipids from wet microalgae. The optimum conditions maximizing lipid yields were as follows: the proportion of methanol to chloroform of 1:1 and the ratio of total solvent volume to dry biomass of 30 mL/g. Water positively affected lipid extraction efficiency when chloroform, methanol, and water formed a monophasic solution. Finally, the newly-developed method was compared with existing extraction methods.