A sensitive and robust mixed-mode high performance liquid chromatography-tandem mass spectrometry method was developed for the qualitative and quantitative determination of sugar phosphates, which are notoriously difficult to separate using reversed-phase materials. Sugar phosphates were separated on a Primesep SB column by gradient elution using aqueous ammonium formate and acetonitrile as mobile phases. Target analytes were identified by their precursor/product ions and retention times. Quantitative analysis was performed in negative ionization/multiple reaction monitoring mode with five different time segments. The method was validated by spiking authentic sugar phosphate standards into complex plant tissue extracts. Standard curves of neat authentic standards and spiked extracts were generated for concentrations in the low picomole to nanomole range, with correlation coefficients of R(2) > 0.991, and the degree of ion suppression in the presence of a plant matrix was calculated for each analyte. Analyte recoveries, which were determined by including known quantities of authentic standards in the sugar phosphate extraction protocol, ranged from 40.0% to 57.4%. The analytical reproducibility was assessed by determining the coefficient of variance based on repeated extractions/measurements (<20%). The utility of our method is demonstrated with two types of applications: profiling of Calvin cycle intermediates in (i) dark-adapted and light-treated tobacco leaves, and in (ii) antisense plants expressing reduced levels of the Calvin cycle enzymes glyceraldehyde-3-phosphate dehydrogenase or ribulose-1,5-bisphosphate carboxylase/oxygenase (comparison with wild-type controls). The broader applicability of our method is illustrated by profiling sugar phosphates extracted from the leaves of five taxonomically diverse plants.