Genus Salmonella of the bacterial family Enterobacteriaceae contains clinically important Salmonella enterica species comprising more than 2600 serovars which cause infections in both humans and animals particularly in poultry, mounting a major public health concern. Due to fecal-oral route transmission of Salmonella, improving sanitation and hygienic conditions can control the disease. However, the target is difficult to achieve in densely populated developing countries like Pakistan, thus precise diagnosis and vaccination are essential. Eleven Salmonella enterica serovar Typhimurium were isolated from 26 Salmonella suspected poultry samples collected from local poultry farms. Different biochemical tests, genus and serovar-specific polymerase chain reactions lead to biochemical identification and molecular confirmation of the isolates. The nucleotide sequencing of the amplified products of 16S rRNA genes resulted in molecular confirmation and NCBI Genbank accession numbers MK041590 and MK041597. Large scale growth of the representative S. Typhimurium MAW1 isolate resulted in sufficient bacterial cell pellet. The purification of lipopolysaccharide (LPS) antigens of the local S. Typhimurium isolate yielded LPS as 53 mg/L of the culture broth. Hyper immune sera were raised in mice by formalin-killed whole-cell bacteria. The LPS endotoxicity was detected as >1000 EU/mL by Limulus Amebocyte Lysate assay and the antigenicity was found adequate by distinct precipitin line against the developed antisera using immuno-diffusion assay. The work will be helpful in making the first polysaccharide-protein based conjugate vaccine candidate against the local S. Typhimurium isolate.