Recent studies have established that dietary protein restriction has beneficial impacts on the metabolic health and it promotes improved glucose homeostasis by increasing the induction of FGF21 from liver. B0AT1 (SLC6A19) is the major neutral amino acid transporter in the intestine that carries out the bulk of amino acid absorption from the diet. It also reabsorbs neutral amino acids in the proximal tubule of the kidney. Mice lacking B0AT1 show signs of protein restriction such as elevated levels of FGF21 and reduced mTORC1 activity. Moreover they have improved glucose homeostasis and are protected from diet induced obesity. Pharmacological blockage of this transporter is expected to replicate this phenotype and it could be employed to treat metabolic diseases such as type 2 diabetes. To evaluate the efficacy of inhibitors of B0AT1, we aimed to develop biomarkers that can reliably detect successful inhibition of B0AT1 in mice in vivo. B0AT1 wildtype and knockout mice served as surrogates for control and fully inhibited B0AT1 activity. Lack of B0AT1 was confirmed by delayed plasma appearance rates of [14C]-labelled neutral amino acids compared to wildtype mice after intragastric administration of a mixture of amino acids. A GC-MS based non targeted metabolomics approach was used to discriminate metabolites profiles in plasma, urine and faecal samples. Lack of B0AT1 was readily detected by significantly increased abundance of neutral amino acids in urine and faecal samples. Metabolites of bacterial protein fermentation such as cresol, 3-indole-propionic acid, 3 hydroxyphenylacetate were more abundant in B0AT1 knock out mice, indicating protein malabsorption of dietary amino acids. ROC curve analysis was used to validate the use of these metabolites as biomarkers. These findings provide putative metabolite biomarkers that can be used to detect protein malabsorption and the inhibition of this transporter in intestine and kidney.