Recombinant protein expression and purification are crucial for studying pathogen–host interactions, especially in therapeutic contexts. This chapter comprehensively overviews various expression systems and chromatography techniques to express and purify respiratory syncytial virus (RSV) proteins. These proteins include nonstructural protein 1 (NS1), nonstructural protein 2 (NS2), nucleoprotein (N), phosphoprotein (P), matrix protein (M), M2–1 protein, M2–2 protein, and the large RNA-dependent RNA polymerase protein (L). Choosing an appropriate expression system, such as bacterial E coli cells or insect cells, is critical for protein expression and will depend on the specific size and properties of the particular protein. Chromatography is a common technique in protein purification that leverages the protein’s binding affinity, charge, hydrophobicity, or size, but it often requires significant optimization. We outline the protocol for expressing RSV proteins and detail the purification procedures using affinity chromatography, ion exchange chromatography, and size exclusion chromatography to achieve optimal purification of these RSV proteins.