The isotopic dimethyl labeling-based quantitative post-translational modification proteomics was applied to study the phosphoproteomic changes associated with the drought responses of two contrasting soybean cultivars. A total of 9,457 non-redundant, unambiguous, label-independent and repeatable phosphopeptides were subsequently identified from six experimental replicates, corresponding to 9,234 of deduced unique phosphoproteins. These soybean proteins contain a total of 20,880 phosphosites, 84.9% of which were found to be novel phosphosites of the soybean phosphoproteome. The overly post-translationally modified proteins is 2.05% of the phosphoproteins identified. Most of these extensively phosphorylated proteins are photoreceptors, mRNA-, histone- and phospholipid-binding as well as serine/threonine/tyrosine protein kinase/phosphatases. The subgroup population distribution of phosphoproteins over the number of the phosphosite of phosphoprotein follows the exponential decay law, Y=4.13e^(-0.098X)-0.04. From 218 significantly regulated unique phosphopeptide groups, 188 significantly regulated phosphoproteins were found, and they are enriched in biological functions of water transport and deprivation, methionine metabolic process, photosynthesis/light reaction, and response to cadmium ion, osmotic stress and ABA under drought treatment. A total of 15 and 20 drought-tolerant cultivar significantly regulated phosphoproteins are transcription factors and protein kinases/phosphatases, respectively. More than 50% of these phosphoproteins are considered to be novel regulatory components, presumably mediating the development of the soybean drought tolerance under water deprivation process. The association of five randomly selected phosphoproteins with the drought response was corroborated with the qRT-PCR method.