Background <p>Plant growth-promoting bacteria (PGPB) must be able to successfully colonize the plant rhizosphere and remain viable after their release into the field to exert beneficial effects on crop production. Therefore, understanding the behavioral dynamics of PGPB in the rhizosphere is a key challenge to evaluate their persistence and fate under field conditions. This study aimed to develop and validate a strain-specific viability quantitative PCR (v-qPCR) assay for monitoring the viable population dynamics of the PGPB <i>B. velezensis</i> PH-023 in the rhizosphere.</p> Results <p>A v-qPCR method was developed for the unambiguous detection and quantification of <i>B. velezensis</i> PH-023 viable cells and spores in the rhizosphere of tomato. The TaqMan-based qPCR assay was designed within a PH-023-specific molecular marker. The nucleic acid-binding dye PMAxx was used in combination with qPCR to selectively amplify DNA of only viable cells. The v-qPCR showed good linearity over a 4-log range and good amplification efficiency. The performance of the v-qPCR method was assessed in the rhizosphere of tomato plants treated with a commercial biostimulant product based on <i>B. velezensis</i> PH-023 under greenhouse conditions and high and stable survival rates of PH-023 were confirmed by v-qPCR. Finally, confocal microscopy of stained roots of tomato plants inoculated with <i>B. velezensis</i> PH-023-based biostimulant product using a viability test showed preliminary evidence of association of the viable inoculated bacteria with the root surface, forming large cell aggregates in comparison with untreated plants.</p> Conclusions <p>This study offers a strategy to develop and validate a strain-specific viability qPCR method for monitoring the viable population of <i>B. velezensis</i> PH-023 and provides evidence for the colonization ability of this strain formulated as a commercial microbial biostimulant in the rhizosphere. The method will allow further studies of ecological fate, including colonization and survival under field conditions, as well as improvements in formulations and delivery strategies for consistent agricultural benefits. It is expected that the development of the v-qPCR presented here provides a reference for similar studies with other beneficial bacterial strains in different environments. As far as we know, the application of strain-specific v-qPCR approaches for the quantification of the viable population of <i>Bacillus</i> sp. strains in soil or rhizosphere samples have not been reported yet.</p>