<p>An&#xa0;active drug delivery vector of Mg-based micromotor is&#xa0;proposed for enhanced intestinal drug mass spectrometry (MS) detection from proof of concept. Taking diabetes as a&#xa0;disease model, insulin nanoparticles (Ins-NPs) were successfully loaded in chitosan (CHI) layer of Mg-based micromotor (Mg/Au/PLGA/CHI@Ins-NPs) due to electrostatic adsorption with PLGA. The penetration ability of micromotors was evaluated on artificial mucin, which is distributed within about 300&#xa0;μm of the mucus. In addition, in vitro drug delivery and retention was carried out on the isolated small intestine of mice; then, the insulin molecule was determined&#xa0;by ultra-performance liquid chromatography-mass spectrometry (UPLC-MS). By overcoming the mucus barrier and enhancing retention in intestine through active transport of micromotor, insulin ions at <i>m/z</i> 963.9443, 1156.3287, and 1445.1592 were detected by UPLC-MS and classified as [Insulin + 6H]⁶⁺, [Insulin + 5H]⁵⁺, and [Insulin + 4H]⁴⁺. Notably, the mass-to-charge ratio of insulin ions was detected only in micromotor drug delivery systems compared to drug-loaded inert particles in the isolated small intestine, attributed to the intensive penetration and retention capability of micromotors. Meanwhile, this Mg-based micromotor exhibited good biocompatibility and was easy to be removed for the UPLC-MS detection sample preparation. Overall, we provide a potential strategy to detect the low content of drugs with UPLC-MS technique by combining with&#xa0;active micromotor and further broadening the sensing application for untethered micromotor.</p> Graphical Abstract <p></p>