In this paper, an experimental validation of an efficient approach to the Fault Detection and Isolation (FDI) of Induction Motor (IM) is proposed. The problem of Inter-turn short circuits (ITSC) in the stator windings is addressed. By introducing fault factors in the IM model an observer-based residual generator is designed, allowing the detection of ITSC in stator windings. The residual generator is built around an extended Kalman Filter (EKF) in order to estimate state variables and fault factors, which permits the evaluation of the severity of the fault. To overcome the problem of tuning the EKF a PSO algorithm is developed. It carries out a heuristic search of the noise matrices by optimizing a cost function. The proposed solution is validated by computer simulations and by real-time implementation on dSPACE 1104 Digital Signal Processor (DSP) test-bench under the healthy and the faulty conditions of IM. To perform tests under faulty conditions, an IM with customized design is built and the stator is rewound permitting to create ITSC. The results reveal the quick detection of the faults, the quantification of its severity and confirm the efficacy of this observer-based FDI algorithm.