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Deficiency of serum magnesium is associated with the incidence of migraine attacks. The present study aimed to evaluate plasma and erythrocyte magnesium levels in a group of patients diagnosed with migraine. Human donors were selected from basic health units (migraine, n = 25) and from a collection and transfusion unit (control, n = 25), both located in the city of Sinop, Brazil. Plasma and erythrocyte magnesium were assessed using flame atomic absorption. Plasma magnesium concentration was significantly lower in the migraine group (0.172 ± 0.018) compared to the control group (0.197 ± 0.020 mg/L), and erythrocyte magnesium concentration was also lower in the migraine group (0.393 ± 0.053 mg/L) compared to the control group (0.432 ± 0.056 mg/L). The method for analysis of magnesium in human plasma and erythrocytes by flame atomic absorption was shown to be in accordance with validation guidelines. This study shows that plasma and erythrocyte magnesium levels were significantly lower in volunteers diagnosed with migraine compared to healthy volunteers. Furthermore, erythrocyte magnesium proved to be a better marker than plasma magnesium for patients with migraine.