Vascular smooth muscle cells (VSMCs), if activated by growth factors as a consequence of vessel injuries, acquire the ability to proliferate and migrate thus contributing to the formation of neointima and atherosclerotic plaque. In this study, a gel-free and label-free proteomic approach was proposed to highlight factors modulated during VSMC activation. Twenty proteins, differentially expressed between quiescent and activated cells, were identified. A constellation of elements, that move together and are closely and functionally related, was visualized. The great majority of them are involved in cell migration and in adhesion formation, suggesting a pivotal role of these protein complexes on the phenotypic modulation. This study represents a first step to ascertain the precise actors of cell activation, their roles and interactions.