<p>Alkynyl steroid hormones (ASHs), synthetic steroid hormones incorporating an alkynyl group, have been discovered to disrupt the endocrine system in humans profoundly. The existence of ASH residues in daily milk consumption might even heighten the risk of cancer for individuals. In this study, we propose a magnetic solid-phase extraction (MSPE) coupled with click chemistry derivatization-based liquid chromatography-mass spectrometry (LC–MS) method for the rapid and highly sensitive determination of ASHs in milk samples. Specifically, Fe₃O₄ magnetic nanoparticles coated with polypyrrole (Fe₃O₄-PPy) were prepared and utilized as the MSPE adsorbent to direct extract ASHs from milk samples. The entire process of MSPE was completed within 6 min. Subsequently, the reagent 4-azido-N-phenylbenzamide (4-ANPA) was employed for derivatizing the extracted ASHs through copper(I)-catalyzed azide-alkyne cycloaddition reaction (CuAAC). The resulting 4-ANPA derivatives of a total of 8 ASHs were then detected using LC–MS. The established MSPE-click derivatization-LC–MS method exhibited satisfactory limits of detection ranging from 8.2 to 53.4 ng/L, good recoveries between 95.4% and 109.2%, as well as intra-day and inter-day precision with relative standard deviations below 11.3%. Overall, our developed method showcased rapid operation, high efficiency, excellent selectivity, and remarkable sensitivity for quantifying ASH levels in milk samples.</p>