Abstract Purpose We hypothesised that plasticity in signal transduction may be a mechanism of drug resistance and tested this hypothesis in the setting of cetuximab resistance in patients with KRAS/NRAS/BRAF^V600 wild-type colorectal cancer (CRC). Methods A multiplex antibody-based platform was used to study simultaneous changes in signal transduction of 55 phospho-proteins in 12 KRAS/NRAS/BRAF^V600 wild-type CRC cell lines (6 cetuximab sensitive versus 6 cetuximab resistant) following 1 and 4 h in vitro cetuximab exposure. We validated our results in CRC patient samples (n = 4) using ex vivo exposure to cetuximab in KRAS/NRAS/BRAF^V600 cells that were immunomagnetically separated from the serous effusions of patients with known cetuximab resistance. Results Differences in levels of phospho-proteins in cetuximab sensitive and resistant cell lines included reductions in phospho-RPS6 and phospho-PRAS40 in cetuximab sensitive, but not cetuximab resistant cell lines at 1 and 4 h, respectively. In addition, phospho-AKT levels were found to be elevated in 3/4 patient samples following ex vivo incubation with cetuximab for 1 h. We further explored these findings by studying the effects of combinations of cetuximab and two PI3K pathway inhibitors in 3 cetuximab resistant cell lines. The addition of PI3K pathway inhibitors to cetuximab led to a significantly higher reduction in colony formation capacity compared to cetuximab alone. Conclusion Our findings suggest activation of the PI3K pathway as a mechanism of cetuximab resistance in KRAS/NRAS/BRAF^V600 wild-type CRC.