Because of the avoidance of first pass metabolic effects due to direct and rapid absorption with improved permeability, intranasal route represents a good alternative for extravascular drug administration. The aim of the study was to investigate the intranasal pharmacokinetics of two anti-migraine drugs (zolmitriptan and eletriptan), using retro-orbital sinus and jugular vein sites sampling. In a parallel study design, healthy male Sprague–Dawley (SD) rats aged between 8 and 12 weeks were divided into groups (n = 4 or 5/group). The animals of individual groups were dosed intranasal (˜ 1.0 mg/kg) and oral doses of 2.1 mg/kg of either zolmitriptan or eletriptan. Serial blood sampling was performed from jugular vein or retro-orbital site and plasma samples were analyzed for drug concentrations using LC-MS/MS assay. Standard pharmacokinetics parameters such as Tmax, Cmax, AUClast, AUC0-inf and T1/2 were calculated and statistics of derived parameters was performed using unpaired t-test. After intranasal dosing, the mean pharmacokinetic parameters Cmax and AUCinf of zolmitriptan/eletriptan showed about 17-fold and 3-5-fold higher values for retro-orbital sampling as compared to the jugular vein sampling site. Whereas after oral administration such parameters derived for both drugs were largely comparable between the two sampling sites and statistically non-significant. In conclusion, the assessment of plasma levels after intranasal administration with retro-orbital sampling would result in spurious and misleading pharmacokinetics.