Neuroblastoma, a prevalent and aggressive childhood cancer, faces a critical lack of effective treatment options. We systematically analyzed small-molecule perturbation gene expression data from the Library of Integrated Network-Based Cellular Signatures (LINCS), aiming to identify drugs with promising with potential for combination therapy. Following this predictive phase, we proceeded to validate our findings through a series of comprehensive assays. To achieve this, we employed an array of advanced techniques: quantitative proteomics with tandem mass tag labeling, western blotting, immunocytochemistry, and migration analysis. This multifaceted approach allowed us to gain a more profound understanding of the therapeutic potential and the cellular dynamics involved in treating neuroblastoma. Our research led us to hypothesize that a synergistic effect could be achieved in neuroblastoma treatment using a combination of two FDA-approved drugs: sirolimus and pyrvinium pamoate. Upon administering this combined therapy, we observed a significant reduction in cell viability, which was also accompanied by decreased mRNA expression. The effectiveness of this combination was further underscored by colony formation assays, which revealed a notably lower survival rate in neuroblastoma cell lines treated with the drug duo, as opposed to those treated with either drug in isolation. Our comprehensive proteomics analysis, encompassing the identification of 20,623 peptides and 3,415 proteins, shed light on the underlying biological processes affected by the therapy.