Both environmental and economic issues are increasingly pushing for the revalorization of agri-food by-products, including those arising from wine industry. Wastes produced from wine-making processes are important sources of biologically active compounds, mainly phenolic acids and flavonoids, which could be re-used for several applications, for example as additive surrogates or new ingredients in foodstuffs and/or pharmaceuticals. Therefore, the development of methods aimed at isolating, characterizing and quantifying molecules present in winery by-products acquires considerable importance in view of their re-utilization on a large scale. In this connection, this study demonstrated that high-performance thin-layer chromatography (HPTLC) and high-performance liquid chromatography with diode array detection (HPLC-DAD) can operate in synergy for the investigation of pomace and seed materials arising from both white and red cultivars of Vitis Vinifera. By virtue of fingerprint profiling, mass spectrometry (MS) interfacing and band comparison method, HPTLC enabled detection and identification of phenolic acids, non-anthocyanic flavonoids and anthocyanins. On the contrary, only anthocyanins could be identified by HPLC-DAD, and their subsequent quantification showed that malvidin-3-O-glucoside (oenin) was the most abundant one. In parallel, HPTLC has allowed to detect and quantify proanthocyanidins (PAC), showing that only catechin was present in the test samples. Both quantitative analytical methods were validated in terms of linearity, detection and quantification limits and precision.