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The building sector represents around 32% of total final energy demand and 40% of primary energy consumption at European level and is thus very energy-intensive. In this context, the Directive 2010/31/EU "Energy Performance of Buildings" (EPBD recast) establishes a comparative methodology framework for the assessment of buildings' energy performance "with a view to achieving cost-optimal levels". The cost optimal analysis leads to a package of energy efficiency measures that minimizes the global cost over a building's lifecycle. Since it is not possible to apply such analysis to each building, a set of reference buildings (RBs) must be defined to represent the national building stock, as previously done but with different targets. It is important to highlight that hospitals are the most energy-intensive type of building, since they require big amounts of energy for air-conditioning in order to maintain an high air quality level also for the working of equipments and diagnostic tools. To reduce hospitals' energy consumptions while satisfying quality standards, new tools are required. Therefore, referring to the legislative framework in force, this PhD thesis illustrates an original methodology to define a hospital reference building (both for new or existing hospitals) and to evaluate, using a multi-objective approach, the optimal energy performance levels related to different refurbishment scenarios. Referring to a fixed location (Naples, 1034 DD), an original methodology to characterize a hospital reference building is illustrated; in more detail two versions of the reference building were characterized, the first one representative of hospitals built between 1991 and 2005 and the second one representative of new buildings. The core of this methodology is the development of a virtual building based on statistical data and, exceptionally, on experts' knowledge and other sources of information. The methodology is organized in different phases: definition of the geometry, subdivision into thermal zones, definition of the architectural envelope, development of the geometric model, systems' characterization, dynamic energy simulation. The analysis of energy performance, using Energy plus, was carried out for both RBs and the primary energy consumptions for all uses are characterized. Referring to different retrofit's scenario an analysis of elements and technologies for the reduction of the energy demand was carried out, with an approach first focused on the detailed study of the single design element and then on the global analysis of the efficacy of "energy efficiency packages" (EEMs). In order to evaluate the optimal EEMs a new methodology was developed for cost-optimal analysis by means of the multi-objective optimization of hospitals energy performance. Finally a dynamic energy analysis of a real hospital case study, representative of the existing stock, was reported in order to compare with the Reference Building' energy performance.