Lipidomics has been proving that membrane lipids play a crucial role in several cell functions and are involved in several pathologies, including cancer. In fact, beyond a scaffold where proteins and other components are embedded, the cell membrane can also act as a barrier or a target for anticancer drugs. From this point of view, the development of new chemotherapeutic agents should also take into account the role of the membrane in their activity. This Review aims to highlight the importance of anticancer drug-membrane interactions as a powerful strategy to improve cancer therapy. Biophysical techniques emerge, therefore, as essential tools to unveil such interactions. Display omitted •Cancer cells present changes in phospholipid constitution, surface charge and membrane fluidity.•The structural, biophysical and functional differences between cancer and normal cell membranes can modulate drug activity.•Mimetic models have been developed to overcome membrane's complexity and used to study anticancer drug-membrane interactions.•Research of new cancer therapies using membrane biophysics may be a path to overcome the problems found in clinical practice.