A specially designed mental task was performed by 16 subjects for frequency domain analysis of skin conductance (SC) to evaluate mental workload. The task was to memorize target letters, detect them within a 4×4 alphabet arrangement, and answer whether the number of targets contained in the arrangement corresponded to a randomly displayed number. As the number of target letters increased, the score for the card-sort NASA Task Load Index (CSTLX) and task performance increased significantly. The traditional parameter for the number of transient wave forms of SC response (SCR) and the height of its wave did not show any significant effect of task difficulty. In addition, SCRs were subjected to Fourier transformation and integration of the spectrum from 0.03 to 0.5 Hz. This frequency domain analysis enabled detection of small differences in mental workload that could not be detected by traditional amplitude domain analysis. Frequency-based analysis enables easy processing of physiological signals and is very effective in evaluating mental stress using SC recorded under actual environmental conditions such as the driving of a vehicle.