Introduction: Tetraspanins are a family of small proteins that cross the membrane four times and form complexes by interacting between themselves and with a variety of transmembrane and cytosolic proteins, building a network of interactions referred to as tetraspanin web or tetraspanin enriched microdomains (TEMs). These domains provide a signaling platform involved in many important cellular functions and malignant processes. Areas covered: The authors describe the methods and the rationale for targeting tetraspanins in the therapy of cancer in this review. Expert opinion: Targeting tetraspanins in cancer may be a promising therapy due to the importance of tetraspanins in several steps of tumor formation, communication with the environment, dissemination, and metastasis.