Five new triterpenoid saponins, heinsiagenin A 3-O-[α-L-rhamnopyranosyl-(1→2)-β-D-glucopyranosyl-(1→2)]-β-D-glucopyranoside (1), heinsiagenin A 3-O-[α-L-rhamnopyranosyl-(1→2)-β-D-glucopyranosyl-(1→2)]-[β-Dglucopyranosyl-(1→4)]-β-D-glucopyranoside (2), 2α-hydroxyheinsiagenin A 3-O-[α-L-rhamnopyranosyl-(1→2)-β-D-glucopyranosyl-(1→2)]-β-D-glucopyranoside (3), 2α-hydroxyheinsiagenin A 3-O-[β-D-glucopyranosyl-(1→2)]-[β-D-glucopyranosyl-(1→4)]-β-D-glucopyranoside (4) and N-(2S, 3R, 4R-3-methyl-4-pentanolid-2-yl)-18-hydroxylanosta-8 (9), 22E, 24E-trien-27-amide-3-O-[α-L-rhamnopyranosyl-(1→2)-β-D-glucopyranosyl-(1→2)]-[β-D-glucopyranosyl-(1→4)]-β-D-glucopyranoside (5) were isolated from the aerial parts of Mussaenda luteola Delile (Rubiaceae). Structural elucidation was based on the analysis of spectroscopic data (1D and 2D NMR) and HR-ESIMS. Compound 1 showed potent antitrypanosomal activity with an IC50 value of 8.80 μM. Compounds 2–4 showed highly potent antitrypanosomal activity with IC50 values ranging between (2.57–2.84 μM) and IC90 values ranging between (3.36–4.35 μM), which are 5 fold greater than the positive control DFMO (IC50 and IC90 values of 13.06 and 28.99 μM, respectively). Compounds 1 and 2 showed moderate affinity to μ-opioid receptors with Ki values of 9.936 μM and 0.872 μM, respectively compared to a Ki value of 1.958 nM for the positive control, naloxone HCl.