For important structures such as nuclear power plants, In-Structure Response Spectrum (ISRS) analysis is essential because it evaluates the safety of equipment and components installed in the structure. Because most structures are asymmetric, the response can be affected by eccentricity. In the case of seismically isolated structures, this effect can be greater due to the difference between the center of mass of the structure and the center of rigidity of the isolator layer. Therefore, eccentricity effects must be considered when designing or evaluating the ISRS of seismically isolated structures. This study investigated the change of the ISRS of an isolated structure by assuming accidental eccentricity. The variables that affect the ISRS of the isolated structure were analyzed to see what additional impact they had due to eccentricity. The ISRS of the seismically isolated structure with eccentricity was amplified more than when there was non-eccentricity, and it was boosted more significantly in specific period ranges depending on the isolator’s initial stiffness and seismic intensity. Finally, whether the displacement requirement of isolators can be applied to the variation of the ISRS due to eccentricity in the design code was also examined.