Multisite protein phosphorylation plays a fundamental role in metabolic regulation. To detect and quantify in vitro kinase phosphorylation activities, we have developed a highly selective liquid chromatography/tandem mass spectrometry (LC-MS/MS) based method using high-resolution multiple reaction monitoring (MRM) on a triple quadrupole mass spectrometer. This method eliminates the need for stable isotope labeling and enables multiparallel kinase target assays. Using these assays, we have made the first observation of in vitro phosphorylation of different trehalose-6-phosphate synthase (TPS) isozymes. TPSs possess putative Ca2+-independent, sucrose non-fermenting 1-related protein kinase 1 (SnRK1) phosphorylation sites. Sixteen synthetic peptides from six different Arabidopsis thaliana TPS isozymes containing the SnRK1 consensus recognition motif were phosphorylated simultaneously in vitro and their phosphorylation dynamics determined. We achieved absolute quantification of TPS peptide phosphorylation by tuning the mass spectrometer to the corresponding synthetic standard phosphopeptides. The selectivity of the mass spectrometer in the MRM mode compensates for the low ionization efficiency of phosphopeptides in the presence of a complex matrix. Results are in close agreement with recent in vivo studies of TPS phosphorylation and regulation and reveal significant differences in the phosphorylation levels of different TPS members within the TPS gene family ranging over three orders of magnitude. Substituting EGTA for CaCl2 in the reaction mixture reduced the formation of some of the phospho-TPS peptides drastically, indicating that Ca2+-dependent kinases are active in the presence of Ca2+-independent SnRKs. This agrees with the proposed overlap of these kinases’ consensus motifs, and enables delineation between Ca2+-independent and Ca2+-dependent phosphorylation. Results demonstrate that multiparallel kinase target assays are sensitive enough to provide evidence for differential multisite phosphorylation of homologous TPS proteins and their highly conserved putative phosphorylation sites.