This study investigates Alternating Current (AC) electroanaesthesia of rainbow trout (Oncorhynchus mykiss) in comparison with MS222 and clove oil, using plasma cortisol and glucose concentrations as stress assessment indicators. A microcontroller-based apparatus was designed and constructed to allow a programmable voltage-time Pulse-Width Modulated (PWM) electrical wave application through 19×20 cm submersible electrodes for 91 sec in a 33 cm long tank to induce loss of equilibrium and immobility with recovery after 52±27 sec. Recovery after 660±102 sec was observed in MS222-anesthetized fish (after induction for 720±72 sec) and a recovery time of 546±102 sec was observed in clove oil-anesthetized fish (after induction for 144±42 sec) both are significantly longer recovery times in comparison with electroanaesthesia (p<0.001). Using direct enzyme-linked immunosorbent assay (ELISA) for cortisol and enzymatic colorimetric assay for glucose assessments at 0, 1, 6, 12 h after each anaesthesia, the anesthetics indicated similar trend of cortisol responses during 12 h of investigation. The dilatory trend of glucose changes and response derived from anesthetics and electricity and its surge at 6 h after anaesthesia (p<0.05) confirmed glucose as a second order indicator of stress responses. Electroanaesthesia is a fast, economic, eco-friendly and safe anaesthetic method provides desirable trout immobility for aquaculture activities.