This paper presents a new method for seismic vulnerability assessment of buildings with reference to their operational limit state. The importance of this kind of evaluation arises from the civil protection necessity that some buildings, considered strategic for seismic emergency management, should retain their functionality also after a destructive earthquake. The method is based on the identification of experimental modal parameters from ambient vibrations measurements. The knowledge of the experimental modes allows to perform a linear spectral analysis computing the maximum structural drifts of the building caused by an assigned earthquake. Operational condition is then evaluated by comparing the maximum building drifts with the reference value assigned by the Italian Technical Code for the operational limit state. The uncertainty about the actual building seismic frequencies, typically significantly lower than the ambient ones, is explicitly taken into account through a probabilistic approach that allows to define for the building the Operational Index together with the Operational Probability Curve. The method is validated with experimental seismic data from a permanently monitored public building: by comparing the probabilistic prediction and the building experimental drifts, resulting from three weak earthquakes, the reliability of the method is confirmed. Finally an application of the method to a strategic building in Italy is presented: all the procedure, from ambient vibrations measurement, to seismic input definition, up to the computation of the Operational Probability Curve is illustrated.