Administration of glyceryl trinitrate (GTN), a donor of nitric oxide, can induce migraine-like attacks in subjects with migraine. Provocation with GTN typically follows a biphasic pattern; it induces immediate headache in subjects with migraine, as well as in healthy controls, whereafter only subjects with migraine may develop a migraine-like headache several hours later. Interestingly, intravenous infusion with prostaglandin-E (PGE) can also provoke a migraine-like headache, but seems to have a more rapid onset compared to GTN. The aim of the study was to shed light on the mechanistic aspect PGE has in migraine attack development. Therefore, PGE plasma levels were measured towards the (pre)ictal state of an attack, which we provoked with GTN. Blood samples from women with migraine (n = 37) and age-matched female controls (n = 25) were obtained before and ∼ 140 min and ∼ 320 min after GTN infusion. PGE levels were measured using liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis. Data was analyzed using a generalized linear mixed-effect model. Immediate headache after GTN infusion occurred in 85 % of migraine participants and in 75 % of controls. A delayed onset migraine-like attack was observed in 82 % of migraine subjects and in none of the controls. PGE levels were not different between the interictal and preictal state ( = 0.527) nor between interictal and ictal state (defined as having migraine-like headache) ( = 0.141). Hence, no evidence was found that a rise in PGE is an essential step in the initiation of GTN-induced migraine-like attacks.