Abstract: The photosynthetic glycerolipids composition of cyanobacteria is crucial to the structural and physiological aspects of cells. We developed a method of electrospray ionization tandem mass spectrometry coupled with ultra-performance liquid chromatography to qualitatively and quantitatively analyze the lipidomes of the Synechocystis sp. PCC 6803 cells. Shotgun analysis identified photosynthetic glycerolipids, including lipid classes and fatty acyl composition within lipids, and exhibited high efficiency, selectivity, sensitivity and mass accuracy, Under different light intensities, the content of MGDG and PG increased by 34.64% and 68.49% after 2 hours exposition, respectively. The MGDG and PG molecules containing de novo synthesized saturated fatty acids increased more rapidly and drastically than highly unsaturated species. The content of DGDG increased by 26.95% after 12 hours under high light, while the content of SQDG gradually declined. The method established in this study provides a powerful analytical tool for dissecting metabolism and physiological functions of glycerolipids in cyanobacteria.