Wheat flour lipids present a complex mixture of components with varying polarities and have been classified as either starch or non-starch lipids, the latter being further subdivided in free and bound lipids depending on the extraction method and solvents used. A novel separation and detection method for wheat lipids in flour and dough that separates both non-polar and polar lipid classes in a single run HPLC analysis using a monolithic silica column and a quaternary gradient and detects them with evaporative light scattering is presented. The method was applied to study in-depth the changes in lipid distribution during bread dough development, thereby monitoring the fate of the different lipid classes. Full dough development was not necessary to decrease the extractability of both non-polar and polar free lipids and increase their levels in the bound lipid extract. The data for the first time also showed which specific lipid classes were redistributed and transferred from starch to gluten, as confirmed by analyzing starch fractions. In addition, not all redistributed lipids interacted in a similar way with the gluten proteins. Major changes occurred during the first 30 s of mixing after which slight changes were observed for triacylglycerols, free fatty acids, lysophosphatidylcholine, and N-acylphosphatidylethanolamine.