The PI3K⁄AKT⁄mTOR pathway mediates signals for cell growth and survival, transcription and translation, and regulated cell death. The significance of mTOR has been underscored recently by the identification of two distinct cellular complexes: mTORC1 (includes raptor and is rapamycin sensitive) and mTORC2 (includes rictor and is insensitive to rapamycin). Activation of the pathway by insulin (or other mitogens) leads to the phosphorylation of programmed cell death protein 4 (PDCD4) via mTORC1 (indirectly through the ribosomal protein p70 S6 kinase). LanthaScreen™ PDCD4 HEK293E is a human cell line which constitutively expresses GFP-PDCD4 fusion proteins. This kinase target was introduced using lipid transfection followed by selection with Blasticidin. These cells are a clonal population isolated by FACS using GFP fluorescence as a sorting marker. A homogenous immunoassay ('addition only', no wash steps) was developed in 384-well format using a TR FRET-based detection method to monitor the phosphorylation of Ser457 on GFP-PDCD4 using a terbium-labeled phosphospecific antibody. The assay has been optimized for performance under numerous experimental conditions (e.g., cell plating density, DMSO tolerance, insulin stimulation time, etc.) and displays excellent statistical data (Z' > 0.6). Moreover, this cell line has been validated with different stimuli and known inhibitors (shows correct EC50 and IC50 values) and serves as robust cellular readout for mTORC1 signaling.