As one of the main components of the tumor microenvironment (TME), the so-called cancer-associated fibroblasts (CAF) have proven to be a pivotal player in the progression of several solid tumors. CAF are transformed cells that can have origin in multiple cellular types, and are presented as different subsets showing phenotypic heterogeneity and functional diversity. CAF populations extracted from human cancer cells of many types have been shown to possess the ability to influence the known signs of cancer. So far, CAF have been shown to promote tumor cell invasion and migration, angiogenesis, proliferation, adhesion, immunosuppression, treatment resistance and worsen patient’s prognosis. Within the TME, CAF also can be a barrier against drugs and immune responses from the body, contributing to regulate malignant progression. Because of its relevance in the disease mechanisms, efforts should be applied towards the better understanding of CAF features and functions. In that sense, this Methods Collection has the intent to publish novel or improved methods related to CAF cells establishment, culturing, characterization, interactions with tumor cells and other cells from TME, matrix remodeling, and CAF-targeting cancer treatment.