Lactose powder production from whey permeate generates various side-streams. Molecular profiling of these side-streams and lactose powder can help to detect minor compounds affecting lactose crystallization, lactose powder properties and document the composition of the underutilized side-streams. In this study, whey permeate, lactose powder and intermediate streams from trial lactose productions were analyzed using gas chromatography-mass spectrometry (GC–MS) and proton nuclear magnetic resonance (¹H NMR) spectroscopy. In total, 110 compounds were identified and 49 were quantified. Linking the molecular profiles to in-process steps revealed differential compositional attenuation by the unit operations. Small molecules (e.g. methanol) and a few larger molecules (e.g. fatty acids) permeated reverse osmosis membrane, while twenty-three compounds (e.g. hydroxypyruvic acid, malonic acid, gluconic acid and ribonic acid) co-crystallized with lactose and ended up in lactose power. These results help to better understand and control lactose powder production and highlights possibilities to develop new food ingredients.