Though advanced cancers generally display complex molecular portfolios, there is a subset of patients whose malignancies possess only one genomic alteration or alterations in one oncogenic pathway. We assess how N-of-One therapeutic strategies impact outcomes in these patients. From 12/2012 to 9/2018, 429 therapy-evaluable patients with diverse treatment-refractory cancers were presented at Molecular Tumor Boards at Moores Cancer Center at UC San Diego. The clinical benefit rate, defined by RECIST1.1, was assessed for patients with solid tumors who underwent next-generation sequencing (NGS) profiling revealing one genomic or pathway alteration, subsequently managed with N-of-One therapies. Nine of 429 patients (2.1%) met evaluation criteria. Using matched therapy indicated by NGS, the clinical benefit rate (stable disease ≥ 6 months/partial/complete response) was 66.7%. Median progression-free survival was 11.3 months (95% CI: 3.4–not evaluable). Thus, a small subset of diverse cancers has single pathway alterations on NGS testing. These patients may benefit from customized therapeutic matching.