Background Ablation of accessory pathway from the region of anterolateral tricuspid annulus remains the most challenging although multiple approaches have been introduced. Objective To evaluate the feasibility of a sequential supra-to-subvalvular approach for mapping and ablation of right anterolateral accessory pathway. Methods Cases of right anterolateral accessory pathway ablation were reviewed and divided into group A (conventional approach) and B (sequential approach) for comparison. In group B, mapping and initial radiofrequency delivery was performed in the right atrium along tricuspid annulus, with the target marked on the 3D map. Radiofrequency was subsequently applied at the target from the right ventricle with a reversed catheter curve. Procedural and follow-up data were collected from the patients. Results Overall success was achieved in all patients in group B and 13/15 cases in group A. Compared to conventional approach, cases completed with sequential approach had a significantly shorter procedure time (124.5±13.4min vs. 152.1±36.0min, p=0.030) and mapping and ablation time (57.2±16.3min vs. 92.1±40.1min, p=0.016). Radiofrequency time was also shorter in group B but without statistical difference (536.9±161.0 sec vs. 650.7±321.9 sec, p=0.313). Conclusion The sequential approach can be feasible in routine ablation of right anterolateral pathway by improving efficiency during mapping and catheter stability during radiofrequency application.