Protease XIV is the most common used agent for selenium species extraction from biological samples. In this work, the interconversion of selenocystine during extraction with Tris−HCl buffer containing protease XIV was demonstrated. The 29 % of selenocystine was converted to species with retention time corresponding to Se-methyl-selenocysteine. In addition, selenomethionine, selenoethionine, and Se-methyl-selenocysteine were oxidized to their oxo-analogues when 0.1 % Triton X-100 or 0.1 % sodium dodecyl sulphate was used for improvement of extraction efficiency. The interconversion of these species was suppressed by using a protease XXIII, which application for the extraction of selenium species is described here for the first time. The method for speciation analysis of nine Se species (selenate, selenite, selenomethionine, selenocystine, Se-methyl-selenocysteine, selenoethionine, trimethylselenonium, methylselenocysteine oxide, and selenomethionine Se-oxide) using extraction with protease XXIII was developed and validated (limits of quantitation 0.21–0.70 μg/L Se, linearity up to 1000 μg/L Se, spike recovery 92.3–110.1 %). It has also been shown that the extraction efficiency of protease XXIII (average 53 %) is only slightly lower in comparison to protease XIV (average 65 %). The main Se species in biological samples, selenomethionine, selenocystine, and Se-methyl-selenocysteine, were stable in extract for three days at room temperature.