Quantification of lactose in lactose-free products requires a sensitive method and is often hindered by high levels of glucose and galactose that are present after lactase treatment. The enzymatic method presented here includes an enzymatic glucose depletion step and enables sensitive analysis of low levels of lactose at a detection limit of 25 mg kg-1, with repeatability of 57 mg kg-1 and a recovery rate of 94%. This method was used to measure lactose levels in different cheese types and residual levels of lactose in lactose-free products. Lactose concentrations were below the detection limit in all ripened cheeses, below 0.1% in lactose-free products, and highly variable in fresh cheeses and other products.