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To enable reliable quantification of natamycin in rabbit and human plasma, a validated, sensitive and selective liquid chromatographyâtandem mass spectrometry assay was developed. The chromatographic separation was achieved isocratically on a Cyano column using methanol: aqueous 3.5 mM ammonium acetate (pH 4) (90:10 v/v). The assay was validated over a concentration range of 6.25â400 ng/mL with lower limit of detection of 3.12 ng/mL. Quantification was performed using the transitions 664.5â137.2m/z for natamycin and 923.5â183.4m/z for the IS. The method was validated with respect to linearity, accuracy, precision, recovery and stability. This assay has been successfully applied to a pharmacokinetic study of natamycin in NZ rabbit and plasma protein binding in human plasma. Keywords: Natamycin, LCâMS/MS, Pharmacokinetics, Protein binding