Manipulating neural activity is crucial for studying the neural connectivity and the pathophysiology of neurodegenerative disease. Among various techniques for neural activation, direct optical stimulation method with femtosecond-pulsed laser is simple and can be specifically applied on a single neuron. Brief irradiation of femtosecond laser pulses on a neuron elevates intracellular calcium, and it propagates to adjacent neurons. However, the mechanisms of laser-induced neural activation are still unclear. In this report, we have elucidated the mechanism of laser-induced neural activation which could be mediated by superoxide, specifically blocked by diphenyleneiodonium chloride, and depletion in intracellular calcium storage. Furthermore, we also showed that the propagation of calcium initiated by laser stimulation is dependent on the presence of extracellular calcium as well as electrical and chemical synapses. We verified the applicability of such mechanism for the assessment of neuronal functionality, by measuring calcium elevation, intracellular calcium propagation, ROS increase, and performing cell death assay in vehicle and A-treated neurons. This work suggests promising applications of the potential for implementing such laser-induced neural activation for rapid and reliable drug screening.