Oxidative cleavage of carotenoids leads to dialdehydes (diapocarotenoids, DIALs), besides the widely known apocarotenoids. DIALs are biologically active compounds that presumably impact human health and play different roles in plant development and carotenoid-metabolism. However, detection of DIALs in plants is challenging due to their instability, low-abundance and poor ionization efficiency in mass spectrometry. Here, we developed a solid phase extraction (SPE) and derivatization protocol coupled with ultra-high performance liquid chromatography−mass spectrometry (UHPLC-MS) for quantitative profiling of DIALs. Our method significantly enhances the sensitivity of DIALs detection with detection limit of 0.05 pg/mg dried food materials, allowing unambiguous profiling of 30 endogenous DIALs with C5 to C24 from vegetables. Our work provides a new and efficient approach for determining the content of DIALs from various complex matrices, paving the way for uncovering the functions of DIALs in human health and plant growth and development.