After chloroform extraction, the rapid and sensitive determination of spiramycin and neospiramycin can be performed with AASP-diol clean-up cartridges prior to reversed-phase C18 high-performance liquid chromatography. The limits of quantification of spiramycin in plasma and milk are 0.023 and 0.013 microgram/ml, respectively, and those of neospiramycin, are 0.058 and 0.006 microgram/ml, respectively. Application of the method to the analysis of plasma and milk samples obtained from pharmacokinetic studies is described. Spiramycin has a terminal half-life of 14.27 h in plasma and 34.59 h in milk, while neospiramycin has a half-life of 25.62 h in plasma and 105.85 h in milk.