Next-generation sequencing (NGS) can be used to detect tumor-specific genomic alterations. This retrospective single-center study aims to assess the application of an extensive NGS panel to identify actionable alterations and initiate matched targeted treatment for patients with advanced cancer. We analyzed genomic alterations in solid tumor biopsies from 464 patients with advanced cancer with the Foundation Medicine assay (FoundationOne®CDx). Therapeutic implications were determined using the Memorial Sloan Kettering Precision Oncology Knowledge Base (OncoKB) classification. The FoundationOne®CDx was successfully applied in 464/521 patients (89%). The most common altered genes were TP53 (61%), KRAS (20%), CDKN2A (20%), TERT (16%), and APC (16%). Among the 419 patients with successfully analyzed tumor mutational burden (TMB), 43 patients presented with a high TMB (≥10 mutations/megabase). Out of the 126 patients with an actionable target, 40 patients received matched treatment (32%) of which 17 were within a clinical trial. This study shows that the application of NGS is feasible in an academic center and increases the detection of actionable alterations and identification of patients eligible for targeted treatment or immunotherapy regardless of tumor histology. Strategies such as early referral for NGS, inclusion in clinical (basket) trials, and the development of new targeted drugs are necessary to improve the matched treatment rate.