The NPN compounds from dairy processing side-streams are a promising source for new products. In this study, the NPN profile of lactose production samples was screened using GC–MS and ¹H NMR spectroscopy. These analytical platforms allowed the identification of 35 NPN compounds including, amino acids and derivatives, amino alcohols, organic acids, and other classes. Quantification of the NPN compounds revealed their attenuation by unit operations during a trial lactose production. Urea, ammonia, glycerophosphocholine, creatine, creatinine, orotic acid and choline were the most dominant compounds. Mother liquor concentrate had the highest concentration of NPN, whereas lactose powder had substantial relative amounts of N-acetylglucosamine, phosphocholine and orotic acid. The NPN compounds added up to 57–99% of the total nitrogen, depending on the sample type. The highest nitrogen recovery was found for the reverse osmosis retentate, mother liquid concentrate, wash water and reverse osmosis permeate, whereas the lowest was found for lactose powder.