Carcinomas overexpressing EGFR family receptors are of high clinical importance, because the receptors have prognostic value and are used as molecular targets for anticancer therapy. Insufficient drug efficacy necessitates further in-depth research of the receptor biology and improvement in preclinical stages of drug evaluation. Here, we review the currently used advanced 3D in vitro models of tumors, including tumor spheroids, models in natural and synthetic matrices, tumor organoids and microfluidic-based models, as a potent tool for studying EGFR biology and targeted drug development. We are especially focused on factors that affect the biology of tumor cells, causing modification in the expression and basic phosphorylation of the receptors, crosstalk with other signaling pathways and switch between downstream cascades, resulting ultimately in the resistance to antitumor agents.