<p>Billions of specimens are housed in museum collections, but only a fraction are used in research after accession. The chemical composition of museum specimens, such as the presence of plant secondary metabolites (PSMs), is a useful component of the extended museum specimen concept. However, most of the work investigating chemistry of museum specimens has focused on herbarium specimens and not insects, of which some species may sequester and retain plant secondary metabolites. Also, the preparation techniques involved in entomological collections vary substantially and may influence the ability to detect sequestered compounds. The present work uses two <i>Euphydryas</i> butterfly species, <i>E. phaeton</i> and <i>E. anicia</i> (Nymphalidae), that sequester one group of plant secondary metabolites, iridoid glycosides, to ask two questions about the detection of sequestered compounds in museum specimens: (1) Can sequestered iridoid glycosides be detected in insect museum specimens and if so, how far back in time can we detect them? and (2) Do the preparation techniques used on insect specimens impact sequestered iridoid glycoside content? We detected sequestered compounds in insect museum specimens dating back to 1933. We also found that freshly collected specimens had significantly higher amounts of stored defenses compared to all other treatments. This work highlights the potential for use of museum specimens in chemical ecology and informs collections interested in adding chemical content to their extended museum specimen data.</p>