Recent evidence suggests that signaling responses elicited by G-protein coupled receptors (GPCRs) are more complex than first believed. For instance, stimulation of several GPCRs leads to the transactivation of receptor tyrosine kinase (RTK) signaling pathways. Many groups have studied the role and mechanistic details of RTK-GPCR cross communication, using standard biochemical methods. While these studies have helped to elucidate possible intermediates involved in this complex network, the diversity of cell types and receptors used has often lead to conflicting results and provided little quantitative information.We present a quantitative measurement of RTK transactivation, using spatial intensity distribution analysis (SpIDA). Employing confocal microscopy, spatial intensity histograms are fit using super-Poissonian distributions, yielding molecular parameters such as density and particle quantal brightness. SpIDA provides measurement of particle densities and oligomerization states of receptors using single images as input. Thus processes such as transactivation, which results in changes in the oligomerization state, can be monitored. CHO-K1 cells expressing EGFR-GFP were transfected with various GPCRs including β2-arrestin, neurokinin1, angeotensin and dopamine receptors. We monitored the relative distribution of monomeric and dimeric EGFR in response to GPCR stimulation and obtained dose response curves for different EGFR-GFP:GPCR interactions.To confirm our findings, similar dose response curves were obtained using FLIM-FRET data and negative controls were also measured when activation or transactivation was blocked with a variety of biochemical agents including Rab5 and AG1478.By fitting dose response curves to standard models, key parameters such as D50, and Dmax were obtained. To show that those results were not biased by either cell type choice or overexpression, we studied transactivation in situ in primary cell cultures from mouse brain neurons and quantified dimerization of the BDNF receptor due to both direct and trans-activation via dopamine.