The aim of this study was to compare efficiency of RP-HPLC (Reversed-Phase High-Performance Liquid Chromatography) and LOC (Lab-on-Chip) methods for wheat gluten protein quantification regarding clustering of wheat cultivars according to the genetic similarity (HMW-GS combinations), as well as to explore relations of these two methods to wheat quality parameters. For that purpose, wheat quality parameters (protein content, falling number, wet gluten content, gluten index, Farinograph, Extensograph, and Amylograph), as well as amounts of gliadin and glutenin fractions by RP-HPLC and LOC methods were determined in two different sets of wheat cultivars (Croatian and Serbian). The percentages of gluten proteins and the values of quality parameters were used to characterize the samples by principal component analysis (PCA). Gluten protein quantification performed by method based on the protein fraction separation by molecular weights (LOC) was better for grouping of genetically similar wheat cultivars than quantification of proteins separated by their different solubility in specified solvent gradient (RP-HPLC). LOC method showed higher potential in wheat quality prediction.