BACKGROUND To appreciate vitamin B1 status in normal and pathophysiological states in local ivorian populations, a high-performance liquid chromatographic method for the simultaneous determination of thiamine and its phosphate esters: thiamine monophosphate and thiamine diphsophate or cocarboxylase in whole blood has been developed. METHODS The method involves extraction with diethylether, followed by pre-column alkaline derivatization by bromide cyanogen. The extract was analysed by isocratic reversed-phase isocratic high performance liquid chromatography with fluorescent detection. System suitability tests were applied to assess the method's continuing suitability for use. The main tests of method validation: linearity, precision, accuracy and sensibility were applied to the analytical procedure. RESULTS Thiamine and its esters were eluted within 10 minutes with a good resolution. System suitability tests showed that the chromatographic system was suitable for continuing use. Results of validation tests show the reliability of the method : linearity domain, satisfying precision and accuracy, detection limits between 0.067 pg/l et 0.92 pg/l (0.160 fmol/l et 1.822 fmol/l). CONCLUSIONS The proposed method is suitable for determination of thiamine and its phosphate esters in whole blood.