Antimicrobial resistance (AMR) continues to plague healthcare systems with untreatable infections on the rise. Gram-negative bacteria, such as Acinetobacter baumannii (a critical-priority pathogen), are defined by their highly impermeable cell envelopes. This permeability barrier, coupled with active efflux mechanisms and selective uptake channels prevents the intracellular accumulation of a broad spectrum of antibiotics that would otherwise be effective. Here, we aimed to identify genes that maintain cell envelope integrity by performing transposon-directed insertion-site sequencing (TraDIS) coupled with fluorescence-activated cell sorting (TraDISort) and to assess their contribution to resistance on a panel of antibiotics. A library of over 100,000 unique A. baumannii ATCC 17978 transposon mutants was grown in rich media before treatment with 5 µM SYTOX-Green, a membrane-impermeable dye typically used for staining cells with compromised cell envelope integrity. Fractions of the treated population were physically enriched by fluorescence-activated cell sorting (FACS) into low, medium and high fluorescent populations on a BD FACSymphony S6 cell sorter (~5,000,000 cells per fraction). The difference in mutant population dynamics among these fractions was analysed through TraDIS. We showed that mutants of genes with known and predicted functions involved in the biogenesis of cellular envelope components, such as peptidoglycan, capsule polysaccharides and lipooligosaccharide were significantly enriched in the high fluorescence population while showing marked depletion in the low fluorescence population. We also identified a group of genes with unknown function, which may reveal novel mechanisms in restricting antimicrobial uptake. Interestingly, some of the mutants with higher SYTOX-Green permeability tend to have increased sensitivity to colistin (more antibiotic susceptibility tests are underway). Therefore, TraDISort successfully linked the differential uptake of a permeability-restricted fluorescence compound in a mutant library to known and novel resistance determinants. These findings illustrate TraDISort as an effective systematic approach for studying cell envelopes and identifying associated AMR-conferring genes.