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Derivatization of propofol with 3-bromomethyl-propyphenazone followed by LVC/MS analysis.The derivatized propofol was detectable at attogram level in hair and serum.The LLOQ was 0.01 pg/mL serum (equivalent to 50 ag/μL in injectable solution).The ionization suppression was minimized by deactivating the excess reagent with methanol.The concentration patterns of propofol in rat serum and hair were unmatched.A sensitive, selective and precise liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for determination of propofol (PRO) in rat serum and hair has been developed. 3-Bromomethyl-propyphenazone was used as derivatization reagent forming propofol-methyl-propyphenazone compound. The derivatization reaction was optimized and validated for maximum MS sensitivity. The MS instrumental sensitivity reached to 10 attogram. The serum samples were extracted by using Chromabond C8 columns, while hair samples extracted with methanol. The tendency of volatility of PRO was minimized by adding triethylamine to the extract before the use of nitrogen gas for evaporation of solvent. The limit of quantitation (LLOQ) was 0.01 pg/mL and the assay was linear to 10000 pg/mL. The intra-and inter-day precision (RSD%) ranged from 0.33 to 3.44% while the accuracy (Er%, relative error) were −6.4 to 1.1%. The ionization suppression, due to reagent, was minimized by reacting the excess reagent with methanol, and eluting to waste before MS ionization source (2–4.5 min). The method was successfully applied for detection and determination of PRO in rat serum and hair after 7–28 days from administration of only one dose of propofol (10 mg/kg).