A sensitive, simple, rapid and reliable spectrofluorimetric assay was developed for the assay of definite antimigraine drugs namely; Almotriptan malate, Rizatriptan benzoate, Sumatriptan succinate and Zolmitriptan in their pharmaceutical preparations and biological fluid. The suggested procedure was established on determination of the quenching process resulting from the action of the studied drugs on the native fluorescence of Eosin Y via developing of a binary complex reaction between the cited antimigraine preparations and Eosin Y in 0.2 M acetate buffer (pH.3.5). Under the optimized experimental conditions, the relative fluorescence capacity was determined at λ ex =301.3 nm and λ em =542.8 nm. The calibration graphs were linear through extent from 0.07-1.0, 0.20-1.0, 0.2-1.0 and 0.1-1.0 µg/mL, for Almotriptan malate, Rizatriptan benzoate, Sumatriptan succinate and Zolmitriptan, respectively. The detection limits were 0.019, 0.041, 0.055 and 0.032 µg/mL while quantitation limits were 0.059, 0.125, 0.168 and 0.096 µg/mL for Almotriptan malate, Rizatriptan benzoate, Sumatriptan succinate and Zolmitriptan, respectively. The suggested assay has been validated according to ICH and USP guidelines and favorably has been applied to assay of cited drugs in their dosage forms and content uniformity testing. The high sensitivity of the developed assay allowed quantification of the studied antimigraine drugs in human plasma.