mTOR, an evolutionarily conserved Ser/Thr protein kinase, belongs to the PI3K-related kinase family, which also includes DNA-PKcs, ATM, and ATR. Although other PI3K-related kinase family members have been shown to secure genomic integrity by sensing DNA damage and related stresses, mTOR is known to function as a nutrient and growth factor sensor. mTOR is the catalytic subunit of two distinct multiprotein complexes known as mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). In response to growth factor and nutrient availability, these complexes regulate a variety of cellular processes, such as cell growth, proliferation, and survival by modulating downstream effectors, such as S6K1, 4EBP1, and AKT. Therefore, evaluation of mTOR activity has been a clear readout in order to monitor the physiological status of cells in response to environmental cues. Here, we present the current techniques for the assessment of mTOR kinase activity in different experimental settings.