X-ray was utilized as an ionization source for differential ion mobility spectrometry (DMS) for the first time. The utilization of this ionization source increases the potential of DMS system for on-site based applications. The influence of experimental parameters (e.g. accelerating voltage, filament current, and separation field) on the analysis of model compounds was investigated and discussed. It was found that both the positive and the negative reactive ion peaks [RIP(+) and RIP(−)] formed during X-ray ionization are identical with those observed with the traditional ⁶³Ni radioactive ion source. This is especially notable for RIP(−), because the chemistry provided by other nonradioactive sources in the negative mode is more complicated or even different than that observed with a ⁶³Ni source. Increase of either filament current or accelerating voltage resulted in increased intensity of both RIP(+) and RIP(−). However, because of the materials used for construction of X-ray adapter the maximal level of filament current and accelerating voltage used in this study were limited to 700 mA and 5 kV, respectively. Analytical performance was determined with two model compounds (acetone and methyl salicylate) using X-ray and directly compared to ⁶³Ni ionization source. When X-ray was coupled to DMS, calculated LOD values were found to be within the range of 0.17–1.52 ppb v/v (concentration in the carrier gas). These values are competitive with those calculated for DMS equipped with traditional ⁶³Ni radioactive ionization source. The obtained results are promising enough to ensure the potential of X-ray as ionization source for DMS.