A method based on ESI ion mobility spectrometry as a detection technique after treatment with a molecularly imprinted polymer is described for the analysis of pioglitazone. In addition to the molecularly imprinted polymer separation methodology, the positive ion mobility spectrum and the reduced mobility values for pioglitazone are reported for the first time. The method was exhaustively validated in terms of sensitivity, imprinting factor, enrichment factor, and sorption capacity. A linear dynamic range of 0.10-20.00 μg/mL and an RSD below 6% were obtained for the analysis of this compound. The average recovery for the analysis of spiked samples was calculated to be about 91%. The method was also used to determine pioglitazone in cow plasma, and the results were compared with those obtained using HPLC. The satisfactory results evidence a convenient method for the analysis of the target compound in real samples without using any additional derivatization methods.