While the use of CDK4/6 inhibitors has significantly improved outcomes for patients with ER+/HER2- tumours, understanding the mechanisms responsible for resistance is essential to identify predictive biomarkers and alternative treatment options after tumour progression. To this end, we developed in-vitro models of acquired resistance to palbociclib and abemaciclib using MCF7 and T47D cell lines. Genomic, transcriptomic, and proteomic analyses were used to identify potential actionable molecular alterations in these models. Results show that acquired resistance was associated with dysregulation of multiple signaling pathways, including cyclin D-CDK4/6-RB, EGFR/HER and AKT/mTORC1. Strikingly, acquired resistance across all cell lines was also associated with an upregulated interferon (IFN) response. Expression of an IFN-based gene signature derived from these models was upregulated in breast cancer cell lines and early-stage tumours intrinsically resistant to CDK4/6i, and thus warrants further clinical evaluation as a predictive biomarker of resistance.