Non-enzymatic glycation is a process, which can be best described as a significant posttranslational modification of various proteins. It emerges in hyperglycemic conditions and may have an impact on albumin stability as well as its activity and physical and chemical properties, essentially affecting all its physiological functions. The goal of this research was to answer the following questions: (i) how does the glycation of defatted human serum albumin by glucose−fructose syrup (GFS) alter its tertiary structure; (ii) does palmitic acid (PA), a component of palm oil, affect the in vitro glycation process and cause conformational changes of glycated albumin; and (iii) does PA inhibit the formation of Advanced Glycation End-Products (AGEs)? Therefore, in order to point out differences in the tertiary structure of macromolecules, the absorption and emission of fluorescence spectra and their second derivatives, excitation fluorescence and synchronous spectra, Red-Edge Excitation Shift (REES effect), and the degree of modification of sulfhydryl groups of defatted, non-glycated (HSA) and glycated human serum albumin (gHSA) with GFS and glycated with GFS and PA were investigated. In the present study, it has been confirmed that the glycation of albumin in the presence of GFS and PA causes changes in both HSA and gHSA tertiary structures, respectively. Moreover, palmitic acid, at ratios of 1.5:1 and 3:1 with glycated albumin, does not exhibit inhibition of AGEs formation. This study indicates the fact that the structural changes, especially those of glycated albumin, are important for treatment planning because the type of the interaction between the components and their primary transporter may be altered as the disease progresses or in the elderly.