The IGF-1R⁄PI3K⁄AKT pathway mediates signals for cell growth, cell survival, transcription, translation, and glucose uptake. IGF-1R is a receptor tyrosine kinase (RTK) that resides at the top of the pathway. Upon growth factor binding extracellularly, this RTK undergoes auto-phosphorylation at multiple intracellular tyrosine residues (resulting in pathway activation). The LanthaScreen® IGF-1R GripTite™ (HEK 293 MSR) cellular assay utilizes a human cell line that constitutively expresses IGF-1R fusion proteins with yellow fluorescent protein (YFP). This kinase target was introduced using lipid transfection and these cells are a clonal population isolated by FACS, using YFP fluorescence as a sorting marker and Blasticidin to maintain cells under selection. Using this cell line, a homogenous immunoassay was developed with a time-resolved FRET (TR-FRET) readout in which the insulin-induced phosphorylation of multiple tyrosine residues on IGF-1R is detected in cell lysates using a generic terbium-labeled phosphospecific antibody (Tb-anti-pY20). This cell line has been validated with different stimuli⁄inhibitors and shows correct EC50 ⁄ IC50 values. Moreover, this assay has been optimized for performance under variable experimental conditions (including cell plating density, agonist stimulation time, DMSO tolerance and assay development time) and displays excellent statistical data (Z' > 0.6) and good signal-to-background.