Floor response and design spectra of a museum building before and after isolation are studied; these are used for estimating the response and seismic design of acceleration-sensitive non-structural components. First, two finite-element models of the building before and after seismic isolation are established and verified through ambient vibration tests. Second, seven earthquake motions are chosen to analyse the models; differences between floor response curves and earthquake motions are analysed based on peak acceleration and frequency spectra. Finally, taking the floor response curves as input, the floor acceleration response spectra before and after isolation are calculated, and the design spectra are fitted. Analytical results demonstrate that the dynamic characteristics of the finite-element model agree well with test values, and peak acceleration of the isolated structure is around 75% lower than that of the non-isolated structure. The predominant frequency of the floor response curves is consistent with the natural frequency of the structure. Moreover, the floor acceleration response spectra are influenced by the earthquake motions and the structures, which reflect the characteristics of both. The floor design response spectra are divided into different sections and mathematical formulas are developed. The findings could be applied to the seismic protection design of non-structural components.