A new, rapid and highly sensitive and rapid method was proposed for the simultaneous determination trace levels of vitamin C and folic acid in the human urine samples. The inûuence affecting factors on the UA-DLLME such as type and volume of the dispersive and extraction solvent, pH, ionic strength and temperature of sample solution, sonication condition investigated and optimized. Under the optimized extractions, relative standard deviations (RSD) of the analyses less than 6% (n= 3) and detection limit of 1.65-2.05 ng mL -1 respectively. USAEME -HPLC-UV was successfully applied for the simultaneous determination of trace levels of vitamin C and folic acid in human urine Samples.