A high-performance liquid chromatography–fluorescence detection (HPLC-FLD) method was developed and validated for choline quantification in foods. Samples were extracted by homogenizing in chloroform/methanol/water and hydrolyzing in HCl-acetonitrile. Choline was derivatized using 1-naphthyl isocyanate and quantified by HPLC-fluorescence detection. Average recovery using choline iodide as the standard (n = 6) ranged from 84 ± 6 % for whole-wheat flour to 106 ± 5 % for milk. Recovery after addition of dietary lecithin to two different food matrices faba beans and for whole-wheat flour (n = 6) was 83 ± 5 %. The precision of the analysis (coefficient of variation) ranged from 2 to 13 %. Accuracy was evaluated by analyzing dietary lecithin using HPLC-FLD, liquid chromatography–mass spectrometry, and nuclear magnetic resonance, which across the different methods agreed within 15 %. This method was applied to quantify the choline content in different food matrices, and provides a simple, inexpensive method that could be widely used.