The sexually transmitted infection gonorrhea remains a public health concern for becoming resistant to drug treatments available. The purpose of this study was to evaluate the usefulness of the matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) to identify and cluster Neisseria gonorrhoeae. From a current monitoring in Italy, as part of the European Gonococcal Antimicrobial Surveillance Programme (EURO-GASP), 93 gonococci collected from 2007 to 2012 susceptible (44 isolates) and resistant (49 isolates) to cefixime were selected. Minimum Inhibitory Concentration (MIC) values for cefixime was assessed by Etest carried out in agreement with the manufacturer's instructions and interpreted referring to European Committee on Antimicrobial Susceptibility testing (EUCAST) clinical breakpoints criteria. Data obtained by N. gonorrhoeae multiantigen sequence typing (NG-MAST) and the dendrogram based on the concatenation of porB and tbpB genes were evaluated. MALDI-TOF MS, to reconfirm gonorrhea identification, analyzed single colonies from freshly grown isolates and applied directly on a ground-steel MALDI target plate. For the MALDI-TOF dendrogram cluster analysis, MSPs (Main Spectrum Profile) from each isolate were created acquiring 5000 shots from 10 technical replicates obtained from bacteria extraction.