Carotenoid oxidative cleavage products, apocarotenoids (APOs), are a class of important plant secondary metabolites, which include phytohormones abscisic acid (ABA) and strigolactones (SLs), and growth regulators and signaling molecules such as β-cyclocitral, zaxinone, anchorene, β-apo-11-carotenoids, and retinal. Qualitative and quantitative analysis of these bioactive compounds is crucial for understanding their metabolism and may also enable discovering further regulatory APOs. The state-of-the-art mass spectrometry (MS) technology has advanced the detection of plant APOs; however, it is still challenging to perform an accurate analysis of the low-level phytohormones ABA and SL and the structurally diverse APOs from complex plant matrices. Here, we describe ultrahigh-performance liquid chromatography-MS (UHPLC-MS) methods to determine carotenoid-derived hormones and APOs from plants by integrating ultrasound-assisted extraction and solid-phase extraction. These assays enable an accurate quantification of carotenoid-derived hormones and APOs from plant tissues by using an UHPLC hybrid quadrupole-Orbitrap mass spectrometer. © 2022 Wiley Periodicals LLC.