Start-up and operation of single-stage nitritation-anammox sequencing batch reactors (SBRs) for completely autotrophic nitrogen removal can be challenging and far from trivial. In this study, a step-wise procedure is developed based on stoichiometric analysis of the process performance from nitrogen species measurements to systematically guide start-up and normal operation efforts (instead of trial and error). The procedure is successfully applied to laboratory-scale SBRs for start-up and maintained operation over an 8-month period. This analysis can serve as a strong decision-making tool to take appropriate actions with respect to reactor operation to accelerate start-up or ensure high-rate N removal via the nitritation-anammox pathway.