A method for the determination of a macrolide immunosuppressant, L-732,531, in plasma is described. Plasma samples are extracted using cyano solid phase extraction columns. The extract is analyzed by normal phase high performance liquid chromatography with a cyano column and a mobile phase of hexane/2-propanol/water (780:220:3). Detection is based on fluorescence at an excitation wavelength of 276 nm and an emission wavelength of 330 nm. The limit of quantification of the assay is 1 ng/mL. The assay was validated in the concentration range of 1 – 200 ng/mL when 1 mL aliquots of plasma are extracted.