The advancement of cancer genomics has allowed for multiplex gene assays using next-generation sequencing (NGS) to be practically implemented, however, a clinical practice system remains to be established. We evaluated the feasibility of clinical sequencing using NGS-based multiplex gene assays between cooperating medical institutions in patients with advanced cancers. In this observational study, DNA and RNA samples prepared from existing tumor tissues were subjected to comprehensive genomic profiling using targeted sequencing. From January 2017 to March 2019, 36 samples from 33 patients were assessed. Of all patients, 27 (82%) had lung cancer, with the median age of 50 years (range 38-83). Multiplex gene panel tests were successfully carried out on 35/36 (97%) samples. Potentially actionable gene alterations were identified in 10/30 (33%) samples (3 HER2, 2 KRAS, 2 ALK, 1 PIK3CA, 1 RET, and 1 CDKN2A). In the 6 samples examined for resistant mechanisms, ALK I1171N mutation and MET copy number gain were detected in 2 patients with ALK rearrangement-positive lung cancer. Clinical sequencing using NGS-based multiplex gene assays between collaborating domestic medical institutions was feasible, with a success rate of > 97%. Overall, clinical sequencing benefits therapeutic decision-making in patients with advanced cancer.