Plasma cortisol is the classical parameter to assess acute stress in vertebrates, while in fish larvae whole body cortisol is used. Immuno assays are commonly adopted for cortisol determination in a pool of fish larvae. At present no method exists for quantification of a full glucocorticoid profile of a single fish larva providing a detailed snap-shot of hypothalamic-pituitary-interrenal (HPI) axis activity. To date, a substantial amount of studies has confirmed the potential of probiotics to positively impact fish welfare by alleviating the general stress response. However, no study investigated how probiotics impact the glucocorticoid profile in an individual fish larva. In this respect, this study assessed the effect of the administration of the probiotic candidate Vibrio lentus, previously shown to protect European sea bass (Dicentrarchus labrax) larvae, on the HPI axis activity. Therefore, V. lentus was administered to germ-free European sea bass larvae and a ultra-performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS) method for quantifying cortisol, its precursors (17α-hydroxyprogesterone and 11-deoxycortisol) and phase I metabolites (cortisone, 20β-dihydrocortisone, tetrahydrocortisol and tetrahydrocortisone) in the whole body of a single fish larva was developed in an EN ISO/IEC 17025 regulated environment and validated according the requirements of the Commission Decision No. 2002/657/EC and applied, making this the first study in its kind. In the supplemented larvae, the administration of the probiotic candidate V. lentus significantly decreased glucocorticoid baseline levels. This study provides a better insight into the effect of the probiotic candidate V. lentus on the HPI axis activity in European sea bass larvae.