Background: Bioactive compounds derived from plants have been extensively studied and widely applied in cosmetic and dermatological fields. In particular, plant-derived polyphenols and anthraquinones are known for their antioxidant properties as well as their ability to inhibit elastase enzyme activity. Elastase is a key proteolytic enzyme responsible for degrading elastin, a structural protein essential for maintaining skin elasticity. Excessive elastase activity contributes to skin aging and wrinkle formation. Therefore, the identification of plant-derived compounds capable of interacting with elastase is of significant importance for improving the composition and efficacy of cosmetic products. Aim: To identify bioactive compounds in the extract of Rheum undulatum L. and to investigate their interaction with elastase enzyme activity. Materials and Methods: The root of Rheum undulatum L. was used as the study material. Sample preparation was performed by grinding the plant root followed by extraction using 50% ethanol assisted by an ultrasonic water bath. The obtained extract was evaporated to dryness using a vacuum centrifugation system to remove the solvent completely. A total of 10 mg of the dried extract was dissolved in 100 µL methanol and 900 µL water, then filtered through a 0.1 µm membrane filter. Subsequently, two sample sets were prepared: a control sample and an elastase-incubated sample at a 1:1 ratio. Both were analyzed using liquid chromatography–mass spectrometry (LC-MS). Data processing was performed using Agilent MassHunter Qualitative Analysis software (version 10.0). Results: LC-MS analysis revealed a total of 16 bioactive compounds in the root extract of Rheum undulatum L. Comparative analysis between the control and elastase-treated samples demonstrated a decrease in the peak areas of gluconic acid and citric acid, while an increase in the peak area of emodin was observed. These changes indicate compositional differences between the two sample conditions. Conclusion The LC-MS analysis of Rheum undulatum L. root extract identified multiple bioactive compounds belonging to anthraquinone, phenolic, and flavonoid classes. Variations in peak intensities between control and elastase-treated samples suggest potential interactions between certain compounds and the enzyme. These findings indicate that the root extract contains biologically active constituents capable of modulating elastase activity, supporting its potential application in anti-aging and cosmetic formulations.