Selenium is an essential trace element in human health and therefore its concentration in biological samples (biofluids and tissues) is used as an indicator of health and nutritional status. In humans, selenium's biological activity occurs through the 25 identified selenoproteins. As total selenium concentration encompasses both functional selenoproteins, small selenocompounds and other selenium-binding proteins, selenium speciation, rather than total concentration, is critical in order to assess functional selenium. Previously, quantitative analysis of selenoproteins required laborious techniques that were often slow and costly. However, more recent advancements in tandem mass spectrometry have facilitated the qualitative and quantitative identification of these proteins. In light of the current alternatives for understanding selenium biochemistry, we aim to provide a review of the modern applications of electrospray ionisation mass spectrometry (ESI-MS) as an alternative to inductively coupled plasma mass spectrometry (ICP-MS) for qualitative and quantitative selenium speciation.