Presented here is the comprehensive speciation of organic and inorganic selenium species via liquid chromatography-particle beam/electron impact mass spectrometry (LC-PB/EIMS). Two different matrices (SELM-1 selenium-enriched yeast certified reference material and synthetic urine) were analyzed for the content of methionine and selenium species utilizing this analytical tool. An ion-paring reversed-phase chromatographic separation using a linear gradient of water and methanol (each containing 0.1% trifluoroacetic acid), at 0.9 mL min⁻¹ with an analysis time of less than 20 minute, was employed for the selenium speciation. Electron ionization mass spectra yielded clear and simple fragmentation patterns of the chemical species eluting from the chromatographic column. Quantification by a standard addition method was accomplished for methionine (m/z = 149 Da) and selenomethionine (m/z = 196 Da) with recoveries of 93% (9% RSD, n = 4) and 97% (11% RSD, n = 4), respectively. Absolute limits of detection on the nanogram level were obtained for each of the targeted species.