Purpose <p>The present study aimed to examine the occurrence of cyclotides in <i>Oldenlandia umbellata</i> L. and to characterize peptide constituents within a cyclotide-rich fraction (CRF–OU) using high-resolution LC–MS/MS. Additionally, the biological activity of the CRF–OU was evaluated to obtain preliminary insights into its functional potential.</p> Methods <p>The cyclotide-rich fraction from <i>O. umbellata</i> L. was subjected to reduction and alkylation followed by tryptic digestion before LC–MS/MS analysis to facilitate peptide characterization. Peptide identification was performed by database searching against CyBase using stringent criteria, including precursor mass accuracy within 5 ppm, database sequence matching, and the presence of six conserved cysteine residues characteristic of cyclotides. The cyclotide-rich fraction was further evaluated for antioxidant activity using the DPPH radical scavenging assay, nitric oxide (NO) modulation in LPS-stimulated RAW 264.7 macrophages, and cell viability.</p> Results <p>LC–MS profiling revealed peptide ions within the typical mass range of plant cyclotides (2.8–3.2&#xa0;kDa). Subsequent LC–MS/MS analysis enabled the identification of two peptides corresponding to Kalata B8 and Kalata B2, previously reported in <i>Oldenlandia affinis</i>. The cyclotide-rich fraction exhibited concentration-dependent radical scavenging activity, significantly suppressed nitric oxide production, and maintained high cell viability at the tested concentrations.</p> Conclusion <p>These findings provide molecular evidence for the presence of Kalata-type cyclotides in <i>O. umbellata</i>. The cyclotide-rich fraction demonstrated antioxidant and nitric oxide-modulating activity in vitro, along with favorable cytocompatibility. However, the specific contribution of individual cyclotides requires further investigation through purification and functional characterization of isolated peptides.</p> Graphical Abstract <p></p>