Current analytical methods lack the capacity of simultaneous determination of the content of free choline and phosphatidyl-bound choline, mainly phosphatidyl choline, in raw milk. Quantitative determination of total, free, and phosphatidyl-bound choline in milk and a dietary supplement is described using a phospholipase D packed bioreactor coupled to a choline oxidase-based amperometric biosensor. The response for choline and phosphatidyl choline was linear up to 0.5 mM and 1 mM, respectively, and the detection limits were 0.02 and 0.03 mM, respectively. The conversion efficiency of phosphatidyl choline to choline was 50% at 0.2 mL min(-1). The within days coefficient of variation for choline and phosphatidyl choline determination in milk samples was 2.8% and 3.2%, respectively. With the addition of an acid hydrolysis step, the method can quantify the concentrations of total, free, phosphatidyl-bound, and non-phosphatidyl-bound choline esters, thus permitting determination of major choline fractions in a complex matrix.