A platform using reverse-phase liquid chromatography coupled to tandem mass spectrometry was developed to measure 28 metabolites from photosynthetic metabolism. It was validated by comparison with authentic standards, with a requirement for distinct and clearly separated peaks, high sensitivity and repeatability in Arabidopsis rosette extracts. The recovery of authentic standards added to the plant material before extraction was 80–120%, demonstrating the reliability of the extraction and analytic procedures. Some metabolites could not be reliably measured, and were extracted and determined by other methods. Measurements of 37 metabolites in Arabidopsis rosettes after 15 min of illumination at different CO2 concentrations showed that most Calvin cycle intermediates remain unaltered, or decrease only slightly (<30%), at compensation point CO2, whereas dedicated metabolites in end-product synthesis pathways decrease strongly. The inhibition of end-product synthesis allows high levels of metabolites to be retained in the Calvin cycle to support a rapid cycle with photorespiration.