The mammalian target of rapamycin (mTOR) is the catalytic subunit of two multiprotein complexes, mTOR complex-1 (mTORC1) and mTOR complex-2 (mTORC2). Clinically used rapamycin and rapalogs are FKBP12-dependent allosteric inhibitors of mTORC1. The recently discovered WYE-125132 and related drugs represent a new generation of ATP competitive and highly specific inhibitors targeting mTOR globally. As mTORC1 and mTORC2 mediate diverse sets of both redundant and distinctive cellular pathways of growth, nutrient and energy homeostasis, rapamycin and WYE-125132 elicit both overlapping and distinctive pharmacological properties with important implications in treating cancer, metabolic, and age-related degenerative diseases. Detailed methods are described for the determination of mTOR inhibition by rapamycin and WYE-125132 in assays of recombinant mTOR enzyme, immunprecipitated native mTOR complexes, growth factor- and amino acid-induced cellular phosphorylation cascades as well as the PI3K/AKT/mTOR hyperactive breast tumor model in vitro and in vivo. The methods have been particularly useful in discovery and biochemical characterization of mTOR inhibitors, cellular and in vivo mTOR substrate phosphorylation analysis, and in deciphering novel biomarkers of mTORC1 and mTORC2 signaling pathways.