This short communication describes the different sample preparation methods for the determination of total selenium in plasma via a spectrophometric method with thionine dye. The blood samples were studied by direct dilution assay with various acid treatments (HCl, HClO₄) and an enzyme treatment (pronase) method. All the samples were analyzed spectrophotometrically with thionine in the presence of KI in HCl medium at 600 nm. The procedure has been validated for estimination of selenium in pharmaceutical tablets. Data showed that pronase treatment is better than the direct dilution method while using thionine dye assay. The advantage of the thionine assay for plasma sample is that it is a simple and rapid technique and can be studied at basic laboratory conditions.