Rapid induction of lipid accumulation in microalgae is an important prerequisite towards the use of microalgae as a feedstock for biodiesel production. In this study, we present a novel approach to induce lipids in Chlorella sp. within 24 h by short-term UV-C radiation (UVR) stress at different energy intensities ranging from 0 to 1000 mJ/cm2. Increase in the lipid fluorescence was measured by Nile red staining and fluorescence-activated cell sorting analysis followed by gas chromatography-mass spectrometry. Lipid fluorescence was significantly increased in cultures radiated at or above 250 mJ/cm2 compared to the mock-treated control cultures. Lower dosages at 100 and 250 mJ/cm2 led to a near doubling of total fatty acids, with a significant increase in unsaturated fatty acids and also most saturated fatty acids. This study provides a protocol for rapid lipid induction of microalgal cells by UV-C and the possible impact of UV-C radiation on fatty acid metabolism.