The scope of this thesis has focused on the investigation of the performance and robustness of UHPSFC-MS in routine laboratories. Furthermore, the analysis of highly polar compounds was tested using UHPSFC, to verify the potential attractiveness of this technique not only as an alternative to RPLC but also to HILIC analysis, in which strongly polar and hydrophilic substances are analyzed. To this purpose, UHPSFC has been applied in different fields, such as untargeted metabolomics employing different biological matrices and peptide analysis. Finally, some fundamental aspects of UHPSFC have been also the subject of work, in an attempt to complement the knowledge on the mechanisms controlling this technique.