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Fentanyl is an opioid analgesic, which is commonly used to treat break through pain in cancer patients or as a pre-procedure anesthetic agent.Recently, mortality cases were reported due to co-administration of fentanyl and other SSRIs like fluoxetine, which is proposed to have metabolic interaction by a liver enzyme CYP3A.A new HPLC-UV method coupled with simple liquid-liquid extraction was developed for fentanyl determination in rat plasma and utilized to elucidate pharmacokinetic behavior of fentanyl after a pre-administration of fluoxetine.