Background: HER2 over-expression or amplification is seen in 4%-16% of BTCs and is a therapeutic target of interest. Gemcitabine-cisplatin (GC) is one of the standard regimens of choice in advanced BTCs. We aimed to evaluate the clinical activity of GC plus anti-HER2 antibody trastuzumab as initial treatment in HER2-positive BTC. Methods: This study was an investigator-initiated, open-label, single-arm, multi institutional, phase II trial in patients (pts) aged 18 years or older with HER2-positive (defined as IHC 3+ or IHC 2+ and FISH positive), treatment naïve BTCs. Patients received GC (Gem 1000 mg/m2 IV and Cisplatin 25mg/m2 IV on day 1 and 8, q 3 weeks) combined with Trastuzumab at 8mg/kg as initial dose followed by 6mg/kg q 3 -weekly till disease progression (PD) or unacceptable toxicities. The primary end-point of the study was an improvement in 6-month progression free survival (PFS) from 40% (historical cohort) to 60% in the study arm. Next generation sequencing to evaluate HER2 and other mutations was conducted on the tissue samples of the patients screened for this study. Results: From March 2020 to August 2022, of the 876 patients were screened for HER2 status, 118 (13.4%) were found to have HER2 positive status and 90 of them were enrolled into the study. A majority of patients had GBC (95.6%) and at-least 2 sites of metastatic disease beyond primary (77.8%). With a median follow up of 8.5 months, 72 patients had PD and the median PFS was 7.95 months (95% CI: 6.84 -9.06; median overall survival (OS) was 9.95 months (95% CI: 9.25 – 10.66). Partial responses (PR) were seen in 8 patients (8.9%) and 61 had stable disease (67.7%) for a disease control rate of 76.6%. Common chemotherapy-related grade 3 or 4 adverse events were anemia [26 (28.9%)], neutropenia [18(20%)] and thrombocytopenia [8(8.9%)] while one patient has Trastuzumab related asymptomatic fall in cardiac ejection fraction (>=10%). The presence of isolated TP53 mutations predicted for inferior PFS compared to patients with other mutations (TERT promoter, HER2, PIK3CA etc) or no detected mutations (6.51 months vs. 12.02 months vs. 10.58 months; p<0.001). Conclusions: The combination of GC and trastuzumab shows near doubling of survival in treatment naive HER2 positive BTC in a prospective manner for the first time and sets the stage for a larger phase III trial with the combination. Evaluating mutations like TP53, HER2, TERT promoter and PIK3CA along with HER2 testing could be considered as a preferred modality to select these patients for anti HER2 therapy. Clinical trial information: CTRI/2019/11/021955.