PTEN/Akt/mTORC 1&2 signal pathway is a critical regulator of cell survival, cell growth and proliferation, angiogenesis, cellular metabolism, cell migration and invasion and glucose uptake. A number of anticancer drugs in clinical trials are targeting this pathway. In Phase I, we will develop reliable, accurate, quantitative and sensitive multiplexed hybrid ELISA/qPCR assay (ELISA combined with quantitative PCR) to accurately monitor protein level and post-translational modifications of the pathway proteins in cells exposed to anticancer drugs rapamycin and LY294002. The ELISA/qPCR kit will be manufactured and written SOPs will be created. In Phase II, we will further improve the assay and use it to quantify the number of target molecules containing important combinations of post-translational modifications in clinical samples of a wide range of cancers. The availability of a sensitive, quantitative and standardized multiplexed ELISA/qPCR will aid clinicians and researchers to profile PTEN/Akt pathway protein members in a number of diseases and predict the efficacy of cancer treatment.