Application of ion mobility spectrometry (IMS) as the detection technique for separation method based on molecular imprinted polymer (MIP) was investigated and evaluated for the first time. Based on the results obtained in this work, MIP-IMS system can be used as a powerful technique for separation, preconcentartion, and detection of metronidazole drug in pharmaceutical and human serum samples. The method is exhaustively validated in terms of sensitivity, selectivity, recovery, reproducibility, and column capacity. The linear dynamic range of 0.05–70.00 g/mL was obtained for the determination of metronidazole with IMS. The recovery of analyzed drug was calculated to be above 89% and the relative standard deviation (RSD) was lower than 6% for all experiments. Various real samples were analyzed with the coupled techniques, and the results obtained revealed the efficient clean-up of the samples using MIP separation before the analysis by IMS as a detection technique.