Objectives: The aim of this study was to analyze the repeatability and validity of different methods to determine the anaerobic threshold through a maximal multistage cycling test; Methods: In total, 17 male endurance-trained athletes [7 cyclists and 10 triathletes, age 33.2 ± 6.9 yr, workload at maximal lactate steady state (MLSS_W) 268 ± 27 W] participated in the study. The participants performed a maximal multistage cycling test twice to analyze the repeatability of the anaerobic threshold (AT) using nine different methods. In the remaining sessions, several 20 min constant load tests were performed to determine MLSS_W (gold standard); Results: The workload corresponding to 73% of the maximal power (AT_73Pmax) showed the best repeatability followed by the Dmax method calculated from the blood lactate concentration ([La⁻]) associated with the Minimum Lactate Equivalent and final [La⁻] (Dmax_LE). Validity analyses showed that all AT determined in the present study were strong predictors of MLSS_W, however, AT_73Pmax and the workloads at 1.5 mmol·L⁻¹ above the first lactate threshold significantly underestimated MLSS_W. The use of correction equations for these variables lowered their absolute mean bias to <5 W. Dmax_LE and workload associated with the 86% of the maximal heart rate showed the narrowest limits of agreement to estimate MLSS_W closely followed by corrected AT_73Pmax; Conclusions: AT_73Pmax, using the correction equation and Dmax_LE, stand out as powerful predictors of MLSS_W among the variables analyzed in the present study in trained cyclists or triathletes. Sports physiologists and coaches can use corrected AT_73Pmax and Dmax_LE to accurately assess athletes’ endurance capacity and prescribe their training.

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