An improved and easy to use method for the determination of thiamin diphosphate (TDP) in 100 μl of whole blood was developed. The small sample volume makes it possible to assess the nutritional status of vitamin B 1 in infants and even in preterm infants. Sample preparation comprises the extraction of TDP from whole blood by hemolysis, protein precipitation with trichloroacetic acid, and subsequent centrifugation. Potassium ferricyanide is used for pre-column derivatization of TDP to its fluorescent thiochrome derivative. Chromatographic separation was carried out using a reversed-phase column and an isocratic elution which consisted of a phosphate buffer and acetonitrile. TDP was detected fluorimetrically and quantified by external standardization. Method validation showed a high precision, almost complete recovery, and a high sensitivity. The lower limit of detection and the lower limit of quantification were 0.2 ng/ml and 4 ng/ml, respectively. Linearity was demonstrated over the expected concentration range of 4–400 ng/ml. In conclusion, we present a convenient HPLC method for the determination of TDP which is precise, sensitive and suitable for pediatric diagnostics.