Vitamin E is known as the most important lipid antioxidant and is widely used to prevent age-associated diseases. Despite increasing knowledge about human vitamin E metabolism, little is known to justify its widespread use. As meta-analyses revealed even harmful effects of high vitamin E doses, a profound understanding of vitamin E metabolism is mandatory. By recent advances in analytical methodology, new metabolites with distinct physicochemical and biological properties were discovered. This review covers current methods to analyze vitamin E metabolites in biological samples. Special emphasis is laid on analytical applications for the identification and quantification of metabolites with a modified hydroxychromanol ring or a truncated side chain.