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Background: Bacterial resistance, exacerbated by multidrug-resistant Gram-negative (GN) pathogens, poses a public health threat due to their impermeable envelopes, which block many antibiotics. Objectives: We aimed to develop a high-throughput screening (HTS) method to identify small molecules targeting GN bacterial envelopes and assess their antibacterial potential. Methods: Envelope disruption in Escherichia coli and Pseudomonas aeruginosa was assessed using a beta-galactosidase (LacZ)/CPRG reporter assay in LB at 37 degrees C. The assay was validated through screening the LOPAC1280 and KD24761 compound libraries. Concentration-response relationships, permeabilisation constants (K50), co-permeabilisation assays, minimal inhibitory concentration (MIC) measurements, and bacterial microscopy post-MICs were performed. Results: The assay demonstrated robust performance, evidenced by high Z-prime factor and signal-to-noise (S/N) ratios. Screening identified 57 active compounds (1.2 percent of the library), including beta-lactams and three non-antibiotic molecules-suloctidil, isorotenone, and alexidine-that exhibited concentration-dependent antibacterial activity. Alexidine showed the most potent activity, with the lowest K50 (2.7 x 10^-3 mM) and MICs of 0.004 mM for Escherichia coli and 0.015 mM for Pseudomonas aeruginosa. Suloctidil and isorotenone induced spherical cell morphology, while alexidine induced a filamentous phenotype, indicative of envelope disruption. The assay also identified antibiotics for monotherapy and combination therapy, with ampicillin, alexidine, and suloctidil enhancing chloramphenicol's efficacy against Escherichia coli MG1655. Conclusions: The LacZ/CPRG reporter assay effectively identified compounds targeting bacterial envelopes, including novel molecules with antibacterial activity against GN pathogens, making it a promising tool for antibiotic discovery or combination therapy.