Transformers are one of the most important components of the power system. It is important to maintain and assess the condition. Transformer lifetime depends on the life of its insulation and insulation life is also strongly influenced by moisture in the insulation. Due to importance of this issue, in this paper a new method is introduced for determining the moisture content of the transformer insulation system using dielectric response analysis in the frequency domain based on artificial bee colony algorithm. First, the master curve of dielectric response is modeled. Then, using proposed method the master curve and the measured dielectric response curves are compared. By analyzing the results of the comparison, the moisture content of paper insulation, electrical conductivity of the insulating oil and dielectric model dimensions are determined. Finally, the proposed method is applied to several practical samples to demonstrate its capabilities compared with the well-known conventional method.