Targeted data acquisition using nano-LC coupled mass spectrometry is an emerging approach when there is need to quantify proteins with high accuracy, sensitivity and reproducibility. Nevertheless, creating assays meeting all those criteria still remains a laborious task, especially when investigating low abundant proteins and small concentration changes. In this work we develop a targeted data acquisition workflow reducing time and effort to target and investigate key players of metabolic pathways during the process of adipocyte differentiation. This lead to accurate and sensitive quantification of proteins involved in the synthesis of fatty acids, glycerolipids, glycerophospholipids sphingolipids, the production of energy and reduction equivalents. Additionally low abundant molecules derived from the PPARG and insulin signaling pathway with approximately 400 for the insulin receptor substrate and 1100 copies/cell for PPARγ were determined.