A three-step chromatographic procedure using orthogonal separation mechanisms (size-exclusion, cation-exchange and ion-pairing reversed phase) was developed to purify three low molecular weight selenospecies, including the major compound, from the aqueous extract of monkeypot (Lecythis minor) nuts. The following reversed-phase nanoHPLC-electrospray Q-TOF-MS/MS allowed the formal standardless identification of selenocystathionine and two isoforms of gamma-glutamyl-selenocystathionine. This is the first MS and MS/MS-based formal evidence of the presence of these compounds in a biological sample.