The burden of infections caused by methicillin-resistant Staphylococcus aureus (MRSA) is exacerbated by an increasing prevalence of vancomycin-intermediate S. aureus (VISA) and heterogeneous VISA (hVISA)1, including emerging resistance to alternative antibiotics such as linezolid and daptomycin2. The mechanisms by which MRSA acquires antibiotic resistance in the clinic, however, are not fully understood. We have recently created a series of new semi-synthetic glycopeptide analogues designed to overcome resistance3. Utilizing in vitro resistance induction assays will not only allow us to discern the resistance acquisition in MRSA, but also to unravel the potential modes of action for novel glycopeptides. To do so, MRSA strain (ATCC43300 – vancomycin MIC: 1 µg/mL) was passaged for 20 days in the presence of increasing sub-lethal concentrations of five clinically used antibiotics (vancomycin, daptomycin, dalbavancin, linezolid and cefepime) and three novel next-generation glycopeptide analogues. Day 20 replicates (n=8 per treatment group) were sequenced (Illumina NextSeq) and variants determined via GATK analysis. The results showed that the majority of day 20 replicates exposed to clinically used antibiotics developed a resistant phenotype (MIC ≥2 µg/mL). In contrast, a slow progression in resistance was observed in replicates exposed to dalbavancin and the novel glycopeptides and their MIC values remained below the clinical breakpoint (MIC ≤0.25 µg/mL). Sequencing revealed previously known mutated genes in clinical isolates including walK, agrC, and pbpB genes associated with reduced vancomycin susceptibility, as well as other genomic variants not previously reported. Additionally, mutated mprF and rpoB genes associated with daptomycin resistance were found in daptomycin-induced replicates. Replicates exposed to the three novel glycopeptides exhibited additional genomic variants affecting different pathways, suggesting potential multiple modes of action. These results demonstrated that in vitro induced resistance in MRSA affected similar genes to those identified in clinical isolates, validating that it can predict clinical resistance. The novel glycopeptides, which retain good potency against MRSA, exhibited mutations in more pathways that suggest multiple modes of action compared to clinically used antibiotics.