Abstract Microbiological assays involving an Escherichia coli lysine auxotroph have been developed previously for the estimation of lysine bioavailability. To facilitate routine use of an auxotrophic E. coli lysine mutant for a microbial assay, our objectives in this study were to improve the assay with respect to shortening the assay time via increasing inoculum levels, and examine two different minimal media, a minimal salts medium (M9) and a more extensively supplemented minimal medium (DM). The maximum optical density (MOD) response of the E. coli in M9 medium was significantly (p < 0.05) lower than the MOD in the DM medium. However, the time to reach MOD in M9 medium was significantly (p < 0.05) shortened when compared to the time in the DM medium. The total assay time was also significantly shortened from a mean time 13.98 h to 8.19 h by using higher inoculum levels. The results from the study indicated that the E. coli lysine auxotroph bioassay time is shortened by cultivation of the assay bacteria in a more minimal medium and utilization of a more concentrated inocula.