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Dendrobium nobile (D. nobile), a traditional herb known for its immunomodulatory and neuroprotective properties, contains characteristic alkaloids and sesquiterpene glycosides. While alkaloids have been extensively studied, research on sesquiterpene glycosides remains limited. This study established and validated a UHPLC-Q-Trap-MS/MS method for detecting six sesquiterpene glycosides in D. nobile, applying it to pharmacokinetic and tissue distribution studies in rats following oral administration of the D. nobile aqueous extract. Plasma and tissue samples were prepared using methanol for protein precipitation and separated on a Waters Acquity UPLC BEH C18 column. Quantification was performed using multiple reaction monitoring (MRM) in negative electrospray ionization (ESI) mode. Method validation demonstrated specificity, selectivity, precision, accuracy, stability, matrix effects, and recovery rates meeting the criteria for in vivo drug analysis. Pharmacokinetic results indicated that dendronobiloside A, dendronobiloside C, and dendronobiloside D were rapidly absorbed with low plasma concentrations and quick elimination. In contrast, dendronobiloside E, dendroside G, and dendromoniliside D were rapidly absorbed with higher plasma concentrations but also eliminated quickly. Tissue distribution studies revealed that dendronobiloside A, C, and D were detectable in the heart, liver, spleen, lungs, kidneys, stomach, large intestine, small intestine, thymus, and pancreas, but almost undetectable in the brain. And dendronobiloside E, dendroside G, and dendromoniliside D were detectable in all tissues. Overall, the six sesquiterpene glycosides reached various tissues within 2 h of administration, with distribution levels ranked as follows: small intestine > stomach > large intestine > pancreas > lungs > kidneys > liver > heart > thymus > spleen > brain. These findings provide insights into the immunomodulatory mechanisms of D. nobile sesquiterpene glycosides and inform clinical dosing considerations., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)