Transposon directed insertion site sequencing (TIS) is a high throughput method combining transposon mutagenesis with next generation sequencing. TIS libraries can be used to identify mutations the cause fitness defects under conditions of interest. However, many TIS methodologies require active growth steps, to enrich for actively growing or fit mutants. These methodologies can bias the outputs of such experiments by excluding non-replicating or slow growing mutants. To address this issue, we modified the traditional TIS method using propidium monoazide which differentiates between dead and live cells, similar to propidium iodide, by virtue of compromised cell membranes. We call this method Viability-TIS or Vi-TIS. Using this method, we studied the effect of various concentrations of carbenicillin on the E. coli BW25113 mutant library. Vi-TIS could identify tolerant mutants upon exposure to a double Minimum Inhibitory Concentration (MIC) of carbenicillin, which traditional TIS could not. We verified the relative susceptibility and tolerance of the mutants identified by Vi-TIS by in vitro antibiotic treatment. This is a proof of principle study that demonstrates that the Vi-TIS methodology shows comparable results to the common TIS workflow whilst reducing the time require for the experiment. Therefore, this method could have applications in TIS projects where culturing bacteria post stress exposure could lead to erroneous results, or in complex biofilms set-ups and other conditions with high extracellular DNA.