A new analytic methodology based on ultrasonic-assisted cloud point extraction coupled with high-performance liquid chromatography (UA-CPE-HPLC) was developed and successfully applied to determine vitamin B9 in food samples. Polyethylene glycol 6000 (PEG-6000) was used as nonionic surfactant due to its chemical properties and trade availability. All parameters such as surfactant concentration, ionic strength, and the other conditions were also investigated and optimized. The optimal conditions for UA-CPE method were presented with 2.4 % w/v PEG-6000, 14.6 % w/v Na2SO4, and ultrasonic heating assistance at 40 A degrees C for 20 min. The calibration curve for vitamin B9 was linear ranging from 20 to 800 mu g L-1, with correlation coefficients being 0.9944 determined by a HPLC-diode array detector (DAD) detector. Relative standard deviation for 150 mu g L-1 was calculated as 2.87 %. Recovery values in different concentration levels were in the range of 95.6-104.2 %. The obtained results demonstrate that the proposed method can be applied for determination of vitamin B9 in real samples.