BackgroundNew treatment strategies are required against infections caused byHelicobacter pylori,which grows increasingly resistant to antibiotics. Polymerase chain reaction-based methods for antibiotic susceptibility testing are available for detectingH. pylori-specific mutations that confer resistance to clarithromycin and levofloxacin. Several meta-analyses have compared eradication rates for susceptibility-guided versus empirical therapy forH. pyloritreatment; however, all have significant limitations and high heterogeneity, and the results are contradictory. The main objective of this trial is to assess whether a sequential strategy based on molecular susceptibility testing-guided therapy forH. pylorihas a better eradication rate than empirical therapy.MethodsThis trial is designed as a prospective, randomised, open-label, active-controlled and single-centre study. Men and women who areH. pylori-positive, naïve to treatment, and aged 18–65 years will be recruited. A total of 500 participants will be randomised to receive either empirical therapy or a susceptibility-guided sequential strategy. Bismuth quadruple therapy will be the empirical first-line therapy, and in case of failure, high-dose dual (proton-pump inhibitor + amoxicillin) treatment will be the rescue therapy. For the susceptibility-guided sequential strategy, regimen selection will be based onH. pylorisusceptibility to clarithromycin (first-line) and levofloxacin (rescue). A first-line treatment of clarithromycin triple therapy will be selected for clarithromycin-sensitive strains. For clarithromycin resistance, a high-dose dual therapy will be selected. During the rescue treatment, a levofloxacin quadruple regimen will be selected for levofloxacin-sensitive strains, and a furazolidone quadruple regimen will be selected for others. The primary outcome is the first-line eradication rate in both groups, and the overall (including first and rescue therapies)H. pylorieradication rate in both groups is one of the secondary outcomes. The eradication rates ofH. pyloriwill be analysed by intention-to-treat analysis, modified intention-to-treat analysis, and per-protocol analysis.DiscussionThis randomised controlled trial will provide objective and valid evidence about the value of polymerase chain reaction-based molecular methods for antibiotic susceptibility testing in guidingH. pylorieradication.Trial registrationClinicaltrials.gov NCT05549115. Released on 18 September 2022. First posted on 22 September 2022. Enrolment of the first participant on 20 September 2022. The study is retrospectively registered.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13063-023-07457-z.