The safety assessment of probiotic bacterial strains has become increasingly essential due to potential health concerns linked to their use under various medical conditions. In this study, we applied in vitro methods, in accordance with FAO/WHO and ICMR/DBT guidelines, to assess the safety profile of two native probiotic strains, Lactiplantibacillus plantarum HD48 and HD51 in accordance with the guidelines set by FAO/WHO and ICMR/DBT. Different assays were conducted to evaluate mucin degradation and blood hemolysis activity, antimicrobial susceptibility, the presence of virulence factors and production of biogenic amines. Lactiplantibacillus plantarum HD48 and HD51 were found to be non-hemolytic and non-mucinolytic. High-performance liquid chromatography (HPLC) analysis revealed the strains did not produce biogenic amines, such as cadaverine, histamine, tyramine and putrescine. Lpb. plantarum HD48 and HD51 were found to be susceptible to most of the antibiotics tested, with the exception of resistance to vancomycin and nalidixic acid; HD48 also showed intermediate susceptibility to norfloxacin and chloramphenicol, and HD51 showed intermediate susceptibility to vancomycin and nalidixic acid. Targeted PCR assay confirmed the absence of the screened genes associated with antibiotic resistance and virulence factors. Further, the strains HD48 and HD51 did not cause any significant (P > 0.05) changes in cell viability of Caco-2 cells as determined by the MTT assay (95%-99%). In conclusion, the results indicate a favourable in vitro safety profile of Lpb. plantarum HD48 and HD51. Therefore may be potentially suitable for a range of food applications and collectively these findings suggest that Lactiplantibacillus plantarum HD51 and HD48 are safe for use and warrant further validation through in vivo studies in both healthy and diseased models.