The consumption of poor-quality protein increases the risk of essential amino acid (EAA) deficiency, particularly for lysine, threonine and methionine. Thus, it is necessary to be able to easily detect EAA deficiency. The purpose of this study was to develop metabolomic approaches to identify specific biomarkers for an EAA deficiency. Sixty Wistar Han male rats were fed for three weeks either a control (20% milk protein), or protein deficient (5% milk protein), or lysine or threonine or methionine deficient diets (25% of the EAA requirement, n=10 for each treatment), and were thereafter supplemented over 3 weeks using a control or control-equivalent diet. All the deficient EAA diets were produced using milk protein (5%, to set the level of deficiency) and free crystalline AA (all other AA except that targeted for deficiency, to create an AA-specific deficiency). Markers for growth (body weight, body composition) were measured and a 24h urine sample was collected after each deficiency and supplementation period, and were analysed using LC-MS. Data were analysed by targeted metabolomic and PLS DA (Partial Least Squares Discriminant Analysis) was applied. Each metabolite identified as significant was then tested by a one-way ANOVA to evaluate the diet effect. Results confirmed that protein and EAA deficiency induced growth retardation, and supplementation permitted growth restoration, but a delay in length, lean body mass and specific organ weights remained following supplementation. The urinary metabolome allowed discrimination between the deficient and nondeficient diets, and specific signatures for methionine and lysine deficiency were identified. Further analyses are required to investigate a specific signature for threonine intake. Urinary metabolites from the lysine degradation pathway, particularly pipecolate and N-N-N-Trimethyllysine, demonstrated lysine deficiency. Results showed that EAA deficiencies influenced the urinary metabolome. The urinary biomarkers identified could be easily applied to detect EAA deficiencies or protein status.