Metabolic profiling of various microalga species and their genetic variants, grown under varied environmental conditions, has become critical to accelerate the exploration of phytoplankton biodiversity and biology. The accumulation of valuable metabolites, such as glycerolipids, is also sought in microalgae for biotechnological applications ranging from food, feed, medicine, cosmetics to bioenergy and green chemistry. In this report we describe the direct analysis of metabolites and lipids in small cell populations of the green alga Chlamydomonas reinhardtii, using laser ablation electrospray ionization (LAESI) mass spectrometry (MS) coupled with ion mobility separation (IMS). These microorganisms are capable of redirecting energy storage pathways from starch to neutral lipids depending on environmental conditions and nutrient availability. Metabolite and lipid productions were monitored in wild type (WT), and genetically modified C. reinhardtii strains with an impaired starch pathway. Lipids, such as triacylglycerols (TAG) and diacylglyceryl-N,N,N-trimethylhomoserine (DGTS), were monitored over time under altered light conditions. More than 200 ions related to metabolites, e.g., arginine, cysteine, serine, palmitate, chlorophyll a, chlorophyll b, etc., were detected. The lipid profiles at different light intensities for strains with impaired starch pathway (Sta1 and Sta6) contained 26 glycerolipids, such as DGTS, monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG), as well as 33 TAG species. Results were obtained over a 72 hour time period under high and low light conditions for the WT species and the two mutants. Our results indicate that LAESI-IMS-MS can be utilized for the rapid analysis of increased TAG production at elevated light intensities. Compared to WT, the Sta6 strain showed 2.5 times higher lipid production at 72 hours under high light conditions. The results demonstrate our ability to rapidly observe numerous changes in metabolite and lipid levels in microalgal population. These capabilities are expected to facilitate the exploration of genetically altered microalgal strains for biofuel production.