The regular consumption of flavonoids has been associated with reduced mortality and a decreased risk of cardiovascular diseases. The proanthocyanidins found in plasma are very different from the original flavonoids in food sources. The use of physiologically appropriate conjugates of proanthocyanidins is essential for the in vitro analysis of flavonoid bioactivity. In this study, the effect of different proanthocyanidin-rich extracts, which were obtained from cocoa (CCX), French maritime pine bark (Pycnogenol extract, PYC) and grape seed (GSPE), on lipid homeostasis was evaluated. Hepatic human cells (HepG2 cells) were treated with 25 mg/L of CCX, PYC or GSPE. We also performed in vitro experiments to assess the effect on lipid synthesis that is induced by the bioactive GSPE proanthocyanidins using the physiological metabolites that are present in the serum of GSPE-administered rats. For this, Wistar rats were administered 1 g/kg of GSPE, and serum was collected after 2 h. The semipurified serum of GSPE-administered rats was fully characterized by liquid chromatography tandem triple quadrupole mass spectrometry (LC-QqQ/MS(2)). The lipids studied in the analyses were free cholesterol (FC), cholesterol ester (CE) and triglycerides (TG). All three proanthocyanidin-rich extracts induced a remarkable decrease in the de novo lipid synthesis in HepG2 cells. Moreover, GSPE rat serum metabolites reduced the total percentage of CE, FC and particularly TG; this reduction was significantly higher than that observed in the cells directly treated with GSPE. In conclusion, the bioactivity of the physiological metabolites that are present in the serum of rats after their ingestion of a proanthocyanidin-rich extract was demonstrated in Hep G2 cells.