Antibiotic resistance is a growing threat to public health, making the development of antibiotics of critical importance. One promising class of potential new antibiotics are ribosomally synthesized and post-translationally modified peptides (RiPPs), which include klebsidin, a lasso peptide from Klebsiella pneumoniae that inhibits certain bacterial RNA polymerases. We develop a high-throughput assay based on growth inhibition of Escherichia coli to analyze the mutational tolerance of klebsidin. We transform a library of klebsidin variants into E. coli and use next-generation DNA sequencing to count the frequency of each variant before and after its expression, thereby generating functional scores for 320 of 361 single amino acid changes. We identify multiple positions in the macrocyclic ring and the C-terminal tail region of klebsidin that are intolerant to mutation, as well as positions in the loop region that are highly tolerant to mutation. Characterization of selected peptide variants scored as active reveals that each adopts a threaded lasso conformation; active loop variants applied extracellularly as peptides slow the growth of E. coli and K. pneumoniae. We generate an E. coli strain with a mutation in RNA polymerase that confers resistance to klebsidin and similarly carry out a selection with the klebsidin library. We identify a single variant, klebsidin F9Y, that maintains activity against the resistant E. coli when expressed intracellularly. This finding supports the utility of this method and suggests that comprehensive mutational analysis of lasso peptides can identify unique and potentially improved variants.