We propose a novel technique that constructs gene regulatory networks from DNA microarray data and gene-protein databases and then applies Mason rule to systematically search for the most dominant regulators of the network. The algorithm then recommends the identified dominant regulator genes as the best candidates for future knock-out experiments. Actively choosing the genes for knock-out experiments allows optimal perturbation of the pathway and therefore produces the most informative DNA microarray data for pathway identification purposes. This approach is more practically advantageous in analysis of large pathways where the time and cost of DNA microarray data experiments can be reduced using the proposed optimal experiment design. The proposed method was successfully tested on the galactose regulatory network.