Glycation is a common non-enzymatic reaction between proteins and sugars, which gives rise in the human body to the formation of advanced glycation end products (AGEs). These modifications impacts both extra and intracellular proteins, leading to cells and tissues dysfunctions. In the skin, accumulation of AGEs leads to aesthetic consequences, wrinkles, dark spots and yellowish skin tone, as it can be seen in diabetic patients. Consequently, there is a growing dermatological interest to find compounds able to eliminate AGEs accumulated in skin. In this context, a method has been developed to detect and quantify intracellular glycation in human dermal fibroblasts. After cultivation of fibroblasts, cell lysates were injected in an HPLC system coupled with a fluorescence detector in by-pass mode. The system allows the simultaneous measurement of global AGEs and particular pentosidine amounts using two sets of wavelengths in a single run of 1 min. The immunocytochemistry approach was used to valid the HPLC analysis data. The method developed was able to quantify changes in global AGEs and pentosidine content in cells in response to glyoxal treatment. Fibroblasts treated with 500 μM of glyoxal for 48 h showed a significant 2.3-fold and 2.6-fold increase in the content of AGEs and pentosidine respectively compared to control cells. As an application, a screening of natural extracts have been done and the method allowed identifying extracts able to significantly reduce the amount of pentosidine in fibroblasts (−32%). These extracts act as deglycation agents of interest in the field of dermatology and cosmetology.