<p>Porcine reproductive and respiratory syndrome virus (PRRSV) is a major pathogen that causes significant economic losses in the global swine industry. Its non-structural protein NSP4 is a 3&#xa0;C-like serine protease (3CLSP), which plays a crucial role in viral polyprotein processing and replication. Here, we prepared two monoclonal antibodies (mAbs) against NSP4, which were designated as 7H10 and 1A2. To indentify the epitopes recognized by these mAbs, the NSP4 protein truncations were performed. The results showed that the minimal epitopes recognized by mAb 7H10 and mAb 1A2 were ¹²² VITEAGEL ¹²⁹ and ¹⁸⁹ VPSDLCALLA ¹⁹⁸, respectively. Alanine scanning analysis indicated that Ile¹²³, Thr¹²⁴, Gly¹²⁷, Glu¹²⁸, and Leu¹²⁹ are critical for mAb 7H10 recognition, while Val¹⁸⁹, Pro¹⁹⁰, Ser¹⁹¹, Asp¹⁹², and Leu¹⁹³ are critical for mAb 1A2 recognition. Moreover, these epitopes are highly conserved among PRRSV-2 strains. These findings provide a foundation for further research into the function of NSP4 and the pathogenic mechanisms of PRRSV.</p>