The high transduction efficiency of lentiviral vectors in a wide variety of cells makes them an ideal tool for forward genetics screenings addressing issues of cancer research. Although molecular targeted therapies have provided significant advances in tumor treatment, relapses often occur by the expansion of tumor cell clones carrying mutations that confer resistance. Identification of the culprits of anticancer drug resistance is fundamental for the achievement of long-term response. Here, we developed a new lentiviral vector-based insertional mutagenesis screening to identify genes that confer resistance to clinically relevant targeted anticancer therapies. By applying this genome-wide approach to cell lines representing two subtypes of HER2(+) breast cancer, we identified 62 candidate lapatinib resistance genes. We validated the top ranking genes, i.e., PIK3CA and PIK3CB, by showing that their forced expression confers resistance to lapatinib in vitro and found that their mutation/overexpression is associated to poor prognosis in human breast tumors. Then, we successfully applied this approach to the identification of erlotinib resistance genes in pancreatic cancer, thus showing the intrinsic versatility of the approach. The acquired knowledge can help identifying combinations of targeted drugs to overcome the occurrence of resistance, thus opening new horizons for more effective treatment of tumors.