In high grade astrocytoma, which are particularly aggressive and difficult to treat, interaction between integrin β1 (ITGB1) and the epidermal growth factor receptor (EGFR) causes therapy resistance owed to enhanced activation of the PI3K/Akt survival pathway. The integrin α5 (ITGA5)-ITGB1 dimer also contributes to survival of high-grade gliomas. With a view to eventually developing a predictive histopathological approach based on measuring molecular interaction of integrins and receptor tyrosine kinases using Förster Resonance Energy Transfer (FRET), in the current work we undertook to characterize the interaction between ITGA5 and EGFR using the A172 astrocytoma cell line as a model system. Cells grown to confluence on fibronectin coated coverslips were, after 2 hours of serum depletion, treated with a saturating dose of human epidermal growth factor (EGF) or left untreated. For direct immunofluorescence labeling, Ab11, specific for EGFR, and SNAKA51, selective for the active conformation of integrin α5 were used after fixation with 1% formaldehyde. For measuring FRET in a Zeiss LSM880 confocal microscope, the spectrally corrected intensity-based FRET approach was used, expanded with pixel-by-pixel correction for autofluorescence. For donor and acceptor, we have used Alexa Fluor 546 and 647 conjugated antibodies and measured FRET in both directions between ITGA5 and EGFR to explore possible stochastic effects. Image processing was done in Fiji, microscopic FRET maps were created using the RiFRET plugin created in our lab. FRET efficiency was in the range of 0.3-0.45 depending on labels and treatments. No change was caused by EGF treatment when all pixels in the image with above-background signal for both proteins were averaged. Consequently, we also implemented morphological gating, thresholding on higher intensity spots corresponding to focal adhesions or clustered EGFR and applying these ROI masks to the FRET maps, but also this approach did not show significant changes upon EGF treatment. We conclude that there is significant molecular interaction between active ITGA5 and EGFR, but this is not sensitive to short term EGF signaling, possibly reflecting the relative temporal stability of cell-matrix adhesion structures.