Abstract Background: Antimicrobial resistance (AMR) in Neisseria gonorrhoeae is an area of increasing concern and to date most studies have utilized genomic rather than proteomic analyses. In this study mass spectrometry-based quantitative proteomics was used to examine the 2016 World Health Organisation (WHO) panel of N. gonorrhoeae isolates with completed closed genomic sequences and well defined phenotypic and genotypic antimicrobial resistance patterns, in order to gain a greater understanding of AMR in N. gonorhoeae. Materials/methods: 14 WHO reference strains were propagated in Morse's Defined Medium. Pooled stable isotope labelled lystates were used as internal standard (IS). Protein lysates were mixed with IS, digested with trypsin and fractionated before analyzed by nano-LC/MS/MS. Each experiment was triplicated independently. The susceptible strain WHO F was used as reference to which the proteomic profiles of other strains were compared with. Reproducibility was checked by hierarchical clustering and permutation adjusted t-tests were performed to find proteins with significant fold changes. Results: A robust method has been developed that was able to produce standardized, reproducible protein expression profiles in Neisseria gonorrhoeae reference strains. Reference strains of N. gonorhoeae that have previously been shown to be genetically highly similar can display different proteomic profiles. Proteins involved in efflux pumping and stress responses were overexpressed. MacB (a macrolide efflux pump), was found to be associated with high level azithromycin resistance, as it was ~2-fold upregulated in WHO V (MIC of azithromycin > 256 mg/L). Conclusions: A robust method was developed to study protein expression in N. gonorrhoeae. The proteome profiles could differentiate genetically similar stains. Even in the absence of azithromycin, some of the overexpressed proteins were associated with AMR strains, like efflux pumps and stress responses proteins. This study identified that the MacB efflux pump could be associated with high level azithromycin resistance.