Vaxsafe® ST (strain STM1) is the only live attenuated vaccine registered in Australia for use in the control of Salmonella in poultry. Attenuation was achieved through the inactivation of aroA in the parent strain, Salmonella enterica subsp. enterica serovar Typhimurium strain 82/6915. The attenuation that makes STM1 an ideal vaccine candidate also renders it difficult to reisolate from mixed samples such as chicken faecal or cloacal swab samples. The microbiological protocols, used for Salmonella detection, described in the ISO 6579-1 and EP Monograph 2.6.13 guidelines result in low and inconsistent re-isolation of STM1. Further development and optimisation of these methods was carried out to improve the recovery rate of Vaxsafe® ST. The optimised method was evaluated using mixed samples where faecal material was present to mimic field conditions. Multiple aspects to the ST reisolation method were re-evaluated and optimised to increase Vaxsafe® STM1 recovery from mixed samples with complex and diverse competing flora. Evaluation of several commercially available plating media indicated that the Oxoid™ OSCM media was the best and most specific STM1 reisolation media. The addition of an antifungal-antimicrobial cocktail during pre-enrichment and aromatic supplement combination during selective enrichment led to the improved STM1 reisolation frommixed backgrounds. The optimised method improved growth of pure STM1 and reisolation of the parent strain, but the reisolation rate was reduced in the presence of competing flora, e.g., in chicken faecal material. This reduction in recovery rate is due to the aroA attenuation of STM1, rendering it less able to compete with other faecal flora compared to the wild type parent ST 82/6915.