This paper presents numerical and experimental investigations on damage detection of mono-coupled multistory buildings using natural frequency as only diagnostic parameter. Frequency equation of a mono-coupled multistory building is first derived using the transfer matrix method. Closed-form sensitivity equation is established to relate the relative change in the stiffness of each story to the relative changes in the natural frequencies of the building. Damage detection is then performed using the sensitivity equation with its special features and minimizing the norm of an objective function with an inequality constraint. Numerical and experimental investigations are finally conducted on a mono-coupled 3-story building model as an application of the proposed algorithm, in which the influence of modeling error on the degree of accuracy of damage detection is discussed. A mono-coupled 10-story building is further used to examine the capability of the proposed algorithm against measurement noise and incomplete measured natural frequencies. The results obtained demonstrate that changes in story stiffness can be satisfactorily detected, located, and quantified if all sensitive natural frequencies to damaged stories are available. The proposed damage detection algorithm is not sensitive to measurement noise and modeling error.