Saponins represent specialized (secondary) metabolites primarily sourced from plants, typically characterized by an aglycone component of triterpenoids or steroids, often referred to as sapogenin, coupled with sugar moieties. Their structural intricacy and diversity, along with their manifold pharmacological properties, have garnered significant interest among researchers. Notwithstanding this interest, the study of saponins has been encumbered by challenges in their isolation, purification, and structural characterization. Fortunately, recent advancements in mass spectrometry (MS) coupled with spectral databases have facilitated the development of efficient dereplication tools. In this context, we present a detailed protocol exemplifying the isolation and structural elucidation of saponins derived from two Asteraceae plants, Brachyscome angustifolia and Tripolium pannonicum. This protocol predominantly relies on nuclear magnetic resonance (NMR) spectroscopy, supplemented by dereplication utilizing tandem mass spectrometry (MS/MS)-based molecular networking (MN), which has accelerated the identification and characterization of novel acylated triterpene saponins.