Protein phosphorylation is one of the major pathways used by eukaryotic cells to propagate signals to the final effectors, regulating multiple aspects of the living cell, such as metabolism, growth, differentiation, adhesion, motility, genome stability and death. In this context, tyrosine kinases (TKs) play a central role in signal transduction and their overexpression or disregulated activity has been implicated in tumor onset and malignancy progression. To date, eight TKs inhibitors have been approved by FDA for the treatment of specific tumors. In spite of their efficacy, insurgence of resistance is a common feature after prolonged administration. The selective pressure by these drugs, in fact, induces clonal expansion of subsets of cancer cells harboring TKs mutations, leading to decreased inhibition potency. Alternatively, resistance to TK inhibitors can be acquired through the activation of others, often unrelated, TKs. For this reason, while stringent target selectivity of TKs inhibitors has been always considered a desirable feature in order to limit toxicity, molecules targeting different TKs have been recently shown to be promising anti-cancer agents as well. Understanding the molecular mechanisms that confer resistance to TK inhibitors, through a combination of enzymatic, structural and cellular studies, is essential in the development of second generation inhibitors active also towards drug resistant tumors.