Diel vertical migration (DVM) bioassay-guided Fourier transform infrared spectroscopy can be a prominent non-destructive and innovative approach for ecological studies. During the characterization of the nature of the fish-exuded kairomone, the peak area results from the spectroscopic analysis of the control, fish-conditioned (F) and temperature incubated fish-conditioned (IF) treatments of the DVM bioassays demonstrated that there was a strong correlation between the alterations of the amine N-H, amide II, amide IV and CH3 asymmetric vibrations, suggesting that both N-H and CH3 molecules may be main components of the fish-exuded kairomone cocktail. The IF treatment, which showed similar results with the control treatment, supported that the kairomone is inactivated by bacterial biodegradation. The seasonal variations in the peak areas of the N-H and CH3 bands suggested that DVM response varied seasonally, where migration response developed quickly in warmer seasons. The peak area of the amine N-H band of the F and IF treatments relative to control conditions can be used as an ideal indicator of the absence or presence and the promptness of migration, at all seasons.