Drug resistance that affects patients universally is a major challenge in cancer therapy. The development of drug resistance in cancer cells is a multifactor event, and its process involves numerous mechanisms that allow these cells to evade the effect of treatments. As a result, the need to understand the molecular mechanisms underlying cancer drug sensitivity is imperative. Traditional 2D cell culture systems have been utilized to study drug resistance, but they often fail to mimic the 3D milieu and the architecture of real tissues and cell-cell interactions. As a result of this, 3D cell culture systems are now considered a comprehensive model to study drug resistance in vitro . Cancer cells exhibit an in vivo behavior when grown in a three-dimensional environment and react to therapy more physiologically. In this review, we discuss the relevance of main 3D culture systems in the study of potential approaches to overcome drug resistance and in the identification of personalized drug targets with the aim of developing patient-specific treatment strategies that can be put in place when resistance emerges.