Didanosine is an effective antiviral drug in untreated and antiretroviral therapy-experienced patients with Human Immunodeficiency Virus (HIV).An automated system using on-line solid extraction and High performance Liquid Chromatography (HPLC) with ultraviolet (UV) detection was developed and validated for pharmacokinetic analysis of didanosiae in dog plasma.Modifications were introduced on a previous methodology for simultaneous analysis of antiretroviral drugs in human plasma.Extraction was carried out on C18 cartridges,with high extraction yield as stationary phase,whereas mobile phase consisted of a mixture of 0.02 M potassium phosphate buffer,acetonitrile (KH2PO4:acetonitrile:96∶4,v/v) and 0.5％ (w/v) of heptane sulphonic acid.The pH was adjusted to 6.5 with triethylamine.All samples and standard solutions were chromatographed at 28℃.For an isocratic run,the flux was 1.0 mL/min,detection was at 250nm and injected volume was 20μL.The method was selective and linear for concentrations between 50 and 5000 ng/mL.Drug stability data ranged from 96％ to 98％,and limit of quantification was 25 ng/mL.Extraction yield was up to 95％.Drug stability in dog plasma was kept frozen at -20℃ for one month after thee freeze-thaw cycles,and for 24 h after processing in the auto sampler.Assay was successfully applied to measure didanosine concentrations in plasma dogs.