Lepidium sativum or Hab-el Rashad is extensively cultivated as a food supplement to preserve a healthy state of human considering its nutritional and therapeutical properties. Nutritional aspect as it is a valuable source of proteins, carbohydrates, minerals, and vitamins. Besides, it has a therapeutic aspect, as previous research proved remarkable biological actions, including antidiabetic, antimicrobial, antioxidant, anticancer and numerous health-controlling effects. In the current study, L. sativum seeds were extracted using 70% ethanol and exposed to ultrahigh-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry (UPLC-ESI-MS) to probe its phytoconstituents. This analysis resulted in the annotation of 41 metabolites across several chemical categories, including glucosinolates, alkaloids, flavonoids, fatty acids and hydantoin derivatives. Compounds were annotated by comparing their retention times and fragments with those in an in-house collected chemical compounds database, as well as with data from online dictionaries and previous literature. The drive of this study is to extensively profile the active constituents in L. sativum seeds extract and demonstrate their mass fragmentation patterns in both positive and negative ionization modes.