In present research work, a simultaneous estimation method of Vanillyl mandelic acid (VMA), 5 Hydroxy indole acetic acid (5-HIAA), Homovanillic acid (HVA), 6ß-Hydroxy cortisol using reverse phase HPLC method was developed and validated. Rat 24 hr urine samples were collected and injected directly without extraction, detection was carried out at 235 nm using UV detector. The developed method is précised, accurate and sensitive. Key words:  VMA, HVA, metabolic disorders, RP HPLC method.