Evidence suggests that the increased production of free radicals and reactive oxygen species lead to cellular aging. One of the consequences is lipid peroxidation generating reactive aldehydic products, such as 4-hydroxynonenal (HNE) that modify proteins and form adducts with DNA bases. To prevent damage by HNE, it is metabolized. The primary metabolic products are the glutathione conjugate (GSH-HNE), the corresponding 4-hydroxynonenoic acid (HNA), and the alcohol 1,4-dihydroxynonene (DHN). Since HNE metabolism can potentially change during in vitro aging, cell cultures of primary human dermal fibroblasts from several donors were cultured until senescence. After different time points up to 30 min of incubation with 5 µM HNE, the extracellular medium was analyzed for metabolites via liquid chromatography coupled with electrospray ionization mass spectrometry (LC/ESI-MS). The metabolites appeared in the extracellular medium 5 min after incubation followed by a time-dependent increase. But, the formation of GSH-HNL and GSH-DHN decreased with increasing in vitro age. As a consequence, the HNE levels in the cells increase and there is more protein modification observed. Furthermore, after 3 h of incubation with 5 µM HNE, younger cells showed less proliferative capacity, while in older cells slight increase in the mitotic index was noticed.