Differential scanning fluorimetry (DSF) is a versatile and accessible technique for probing the thermal stability of peptide-major histocompatibility complexes (pMHCs). Understanding the thermal transition midpoint (Tm) between the folded and unfolded states of pMHCs provides critical insights into their structural stability, which is essential for studying antigen presentation and immune responses. pMHC stability is influenced by the inherent structural differences between MHC class I and class II molecules. Notably, class I MHCs require bound peptidesPeptides for stability, while class II MHCs remain stable without them. Class I MHCs typically present peptides of 8–12 amino acids anchored at specific residues, whereas class II MHCs accommodate longer peptides with distinct anchoring positions. This manuscript outlines a comprehensive DSF protocol optimized for pMHCs, highlighting the method’s advantages over other thermal stability techniques, such as differential scanning calorimetry (DSC) and circular dichroism (CD). DSF offers a simpler, cost-effective alternative, utilizing minimal sample volume and readily available real-time PCR (qPCR) instrumentation. We detail critical steps for sample preparation, including optimal buffer selection, dye addition, and degassing procedures, along with specific instrument setup guidelines for both qPCR-based systems and the NanoTemper Prometheus. Data analysis strategies using Microsoft Excel and Origin software are also discussed, including normalization, derivative calculation, and Tm determination. By providing a standardized DSF protocol tailored to pMHC analysis, this manuscript aims to support researchers in efficiently measuring thermal stability, thereby facilitating investigations into pMHC dynamics and immune function.