Cannabis is a plant which contains more than 100 different cannabinoid compounds. CBD is the main non-psychoactive component of the cannabis plant1,2. CBD has antinausea, antipsychotic, pain-relief, anticonvulsive, sleep improvement, anti-inflammatory, antianxiety, and cancer cell antiproliferative effects3. However, the main limitation of CBD is its low absorption capacity in body fluids due to its lipophilic nature4. To overcome this limitation, CBD can be incorporated in drug delivery systems1. In order to evaluate the efficiency of the drug delivery system, it is necessary to study the release kinetics of the active substance using an appropriate analytical method. Therefore, the aim of this study was to develop an ultra-performance liquid chromatography (UPLC) method for CBD determination as well as to validate the method to verify the suitability of the analytical procedure for CBD quantification. To prepare solutions in concentration range from 2.4 µg/ml to 100 µg/ml CBD was dissolved in a mixture of mobile phase solutions a - 0.1% formic acid in water and b - 0.1% formic acid in acetonitrile with a volume ratio of a:b 30:70. To analyze the samples photometric detection (λ = 228 nm) and Waters Acquity UPLC BEH C18, 1.7 µm, 2.1×150 mm column was used. The mobile phase consisted of a:b (v/v) 25:75. Flow rate was set to 0.2 mL/min, column temperature was 30°C ± 5°C and the injection volume was 3 µL. During the validation of the method, series of standard solutions were prepared and analyzed, and the statistical parameters of the obtained calibration curves were evaluated. The obtained results show that CBD detection limit is DL = 0.500 ± 0.041 µg/ml and quantitation limit is QL = 1.514 ± 0.125 µg/ml. The stability of CBD at 37°C is very low, because the CBD degradation is already observed within the first 24 hours. The precision, intermediate precision and total accuracy of the samples are 99.54 ± 0.06%, 99.72 ± 0.10% and 102.36 ± 3.96%, respectively. Robustness was also assessed for the analytical method. It was determined that small variations in analytical conditions do not significantly change the obtained outcome parameters and do not affect the precision of the UPLC method. It was concluded that the UPLC method is suitable for the determination and quantification of CBD and can be used for the determination of CBD release kinetics from liposomes.