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• The features of a real trolley-bus that crosses over the center of Naples are studied. • Electrical measurements were recorded on the actual bus. • The main BESS technologies for the electric vehicles are also presented. • The substitution of the conventional ICE with an on-board BESS is proposed. • To design the unit, a new two steps optimized procedure is proposed and validated. The paper investigates the renewal of a hybrid trolley-bus, powered by a 600 V DC overhead electrical grid. The analysis focuses on replacing the on-board internal combustion engine (ICE) with a battery-based power unit. A novel two-step optimization procedure is proposed for this purpose. The procedure compares the solutions in terms of the total cost sustained by the ownership. By means of an iterative method, the optimal size of the battery unit is designed as a function of power and energy requirements, taking into account cycle life, depth of discharge, working temperature, replacement, and the requirements of the transport service operator. Using the real measurements taken on a trolley-bus operating in the city center of Naples (Italy), several numerical simulations are performed. The simulations examine three alternative Lithium high specific power and energy batteries. The comparison of the results allows to select the best solution among the different technologies for the proposed application. [ABSTRACT FROM AUTHOR]