Purpose: Patients with advanced BTCs have therapeutic options. After progression on gemcitabine and cisplatin, there is no standard of care option available. In our previous phase II trial, we had demonstrated activity with single agent trifluridine/tipiracil, in advanced, refractory BTCs. Here, we determine the safety and efficacy of trifluridine/tipiracil in combination with irinotecan in a phase II trial setting for refractory, advanced unresectable BTCs. Methods: In this single-arm, open-label phase II clinical trial, 28 patients (27 evaluable patients) were enrolled and were treated with: trifluridine/tipiracil is 25 mg/m2 (days 1-5 of 14 day-cycle) and irinotecan 180 mg/m2 (day 1 of the 14-day cycle). The primary endpoint for the study was 16-week PFS rate. 2-stage Simon design was used for early stopping for futility, where 6 or more successes in first 25 evaluable patients would be a successful trial. This study had 80% power to detect a true 16-week PFS rate of 30%, with a 5% significance level when the true 16-week PFS rate is 10%. Patients with BTCs who had progressed on at least one line of systemic therapy were eligible. Pre-treatment biopsies were performed for organoid development. Results: The median age was 68 years and subtypes were intrahepatic (59%), extrahepatic (33%) and gallbladder cancer (8%). No. of prior lines of therapies included: 1 (37%), 2 (41%), ≥3 (22%). 16-week PFS was 37% (10/27; 95% CI: (19-58%)), thereby meeting the criteria for success for the primary endpoint. The ORR and DCR were 20% and 45%, respectively. Median OS was 52.6 weeks (95% CI: 33.6-NE). The most common grade 3/4 toxicities were neutropenia (29%), lymphopenia (15%), hypertension (15%), thrombocytopenia (11%), elevated alkaline phosphatase (11%), fatigue (7%), and diarrhea (7%). 9 out of 27 (33%) and 14/27 (52%) had dose reductions secondary to AEs (cytopenias and fatigue) for trifluridine/tipiracil and irinotecan, respectively and 1 patient discontinued the therapy due to adverse events. Conclusions: The combination of trifluridine/tipiracil plus irinotecan is a safe and efficacious treatment for refractory metastatic and unresectable BTCs. PFS and OS outcomes are promising compared to previous trials in advanced BTCs. A larger randomized trial is needed to confirm these results. This study was approved and funded in part by the NCCN Oncology Research Program from general research support provided by Taiho Oncology, Inc.