<p>The lumpy skin disease (LSD) virus poses a significant threat to bovine health and productivity in endemic regions, highlighting the need for reliable and standardized reagents for serological surveillance. However, the availability of standardized recombinant antigens for serological assays is limited in clinical settings. This study describes a bioinformatics-guided strategy for developing recombinant LSDV P32 and B5R antigens for serodiagnostic applications. Despite their notable immunogenicity, the P32 and B5R proteins, which are hydrophobic envelope proteins with transmembrane regions, present considerable challenges in terms of recombinant expression and purification. Initial attempts to express ectodomain-only constructs (P32_Tr and B5R_Tr) using the pET-33b(+) system resulted in detectable proteins but faced significant challenges during purification. Guided by in silico predictions regarding solubility and aggregation propensity, the expression strategy was modified by employing a thioredoxin fusion system [pET-32a(+)], which facilitated expression and successful purification via Ni-NTA chromatography under denaturing conditions, followed by refolding. The purified recombinant proteins were confirmed using SDS-PAGE and immunoblotting. Preliminary indirect ELISA demonstrated the specific seroreactivity of both P32 and B5R proteins, confirming their antigenicity by showing reactivity with LSDV-positive sera without any cross-reactivity with sera from other bovine viruses. This study establishes a practical workflow to overcome the expression and purification challenges associated with aggregation-prone or hydrophobic proteins, thereby demonstrating the potential of recombinant P32 and B5R proteins as standardized biological reagents for use in serological assays.</p>