An improved UHPLC-DAD based method was developed and validated for quantification of major carotenoids present in spinach, serum, chylomicrons and faeces. Separation was achieved with gradient elution within 12.5 min for 6 dietary carotenoids and the internal standard, echinenone. The proposed method provides, for all standard components, resolution >1.1, linearity covering the target range (R>0.99), LOQ <0.035 mg/L, intra-day and inter-day RSD <2% and 10%, respectively. Suitability of the method was tested on biological matrices. Method precision (RSD%) for carotenoid quantification in serum, chylomicrons and faeces was below 10% for intra- and inter-day analysis, except for lycopene. Method accuracy was consistent with mean recoveries ranging from 78.8-96.9 and 57.2-96.9% for all carotenoids, except for lycopene, in serum and faeces, respectively. Additionally, an inter-laboratory validation study on spinach at two institutions showed no significant differences in lutein or β-carotene content, when evaluated at 4 occasions.