The sensitive identification of molecular markers in complex samples is of foremost importance in several fields. Surface Enhanced Raman Scattering Spectroscopy (SERS) has received increasing interest in recent decades due to its high sensitivity and rapid detection of analytes in low concentration. The project aims at weaving together High-Performance Thin Layer Chromatography (HPTLC) and SERS through a suitable interface in order to expand the applicability of SERS on complex samples. The project breakthrough lies in the development of a substrate, which will take advantage of the chromatographic chemical bridges bonding silica particles with noble metal nanospheres. The interaction of the analyte with the chemical bridges will force the molecules to be in proximity to nanoparticles, providing SERS enhancement. The SERS substrate will also work as HPTLC plate: this is the key to overcome the concept of hyphenation in favor of integration and for the future development of new HPTLC#SERS devices. Main attention will be focused on eliminate the most critical issues of the two techniques - crucial for their actual large scale application. To test the real versatility of the new substrate, comparison with robust techniques will be performed in different fields with fundamental effects on society, as food chemistry, forensics and cultural heritage conservation. In addition, an innovative HPTLC#SERS database will be developed, providing an important deliverable for the characterization of complex matrices; the potential of the new substrates will be tested on several analytes and complex matrices of interest in the three fields. Furthermore, with reference to the great versatility of plasmonic enhancement, the substrate will also be tested for the analysis with Surface-Enhanced InfraRed Absorption (SEIRA), which will represent an alternative detection strategy, amplifying the application potential of the new nano-substrate in the analytical field.