Advanced glycation (AGEs) and lipoxidation (ALEs) products are formed through specific condensation reactions between nucleophiles (amino groups of free amino acids or their residues in peptides, aminophospholipids or proteins) and electrophiles (carbonyls of reducing sugars, oxidized lipids or others) generating well-defined sets of covalent adducts. The ε-amino group of the lysine is the most reactive precursor in proteins and the primary target of carbohydrate attacks. AGEs/ALEs accumulation has consequences in the development of vascular, renal, neural and ocular complications, as well as in the triggering of inflammatory and neurodegenerative diseases. Therefore, AGEs/ALEs detection, quantification and, in some cases, the assessment of the extent of glycation in biomolecules of different matrices represent a factor of primary interest for science. Reliable analytical methods are thus required. Together with basic concepts, this review presents the main advances, challenges and prospects of research involving AGEs and ALEs in biological and food systems, exploring practical strategies to ensure greater reliability in the analysis of these compounds in different matrices.