Background: Estimating the resting motor threshold (RMT) is crucial for both investigative and therapeutic transcranial magnetic stimulation. However, no gold standard exists, as each method involves trade-offs between accuracy, time, and ease of use. This study compared RMT estimates and trial requirements across 4 EMG-based methods—Rossini-Rothwell (RR), maximum likelihood parameter estimation by sequential testing (ML-PEST), Mills-Nithi (MN), supervised parametric estimation (SPE)—and 2 visual methods: RR visual and ML-PEST visual. Methods: In this observational study, RMT was estimated in 20 healthy participants using 6 approaches: 4 EMG-based (RR, ML-PEST, MN, SPE) and 2 visual (RR and ML-PEST). We assessed agreement among EMG-based methods and compared RMT estimates and trial counts across all methods. Results: EMG-based methods showed strong agreement (ICC: 0.97, 95% CI: 0.94-0.99). RMT estimates differed significantly ( P =0.001), with RR Visual yielding the highest values—significantly higher than RR EMG, ML-PEST EMG, and SPE EMG methods. Trial counts also varied ( P <0.001), with ML-PEST requiring the fewest. In subgroup analysis, RR Visual and ML-PEST Visual produced similar RMTs, but ML-PEST Visual needed fewer trials. Conclusions: Adaptive threshold-hunting methods like ML-PEST offer efficient and accurate RMT estimation while reducing the number of required trials, supporting their use in both clinical and research applications.