Antisense PNA-peptide conjugates targeting essential bacterial genes have shown interesting potential for discovery of novel precision antibiotics. In this context, the minimal inhibitory concentration (MIC) assay is used to assess and compare the antimicrobial activity of natural as well as synthetic antimicrobial compounds. Here, we describe the determination of the minimal inhibitory concentration of peptide-PNA conjugates against Escherichia coli. This method can be expanded to include minimal bactericidal concentration (MBC) determination and kill-curve kinetics.