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The Evaluation of mental workload/ performance has usually depended on professionals (captain, pilot) who have a lot of experience on board. We are attempting to evaluate a ship navigator's mental workload based on a physiological index. The physiological indices, heart rate variability (R-R interval), nasal temperature, and salivary amylase, are good indices for reading the mental workload in ship handling. Moreover, we have found a possibility of salivary NO3- as a good index for evaluating the navigator's mental workload. The salivary NO3- is expected to have a specific characteristic to represent quick response on the spot and the trend like moving average. We confirmed the response of students for simulator training, and we were carried out the experiment for professionals on a real ship. This paper proposes that salivary NO3- shows a ship navigator's mental workload for ship handling in the simulator and a real ship. This research, to evaluate the mental workload of navigators using salivary NO3-, is first challenge worldwide.