This chapter focuses on generating macrophages from monocytes and highlights their crucial role in the immune system, particularly their involvement in the pathogenesis of rheumatoid arthritis (RA), the most frequent systemic autoimmune disease. It addresses the limitations of traditional in vitro models, which often rely on high-dose cytokines, and introduces human AB serum as a more physiological option for monocyte-to-macrophage differentiation. Monocyte-derived macrophages (MDMs) by SAB are presented as a relevant model for studying macrophages biology and function. A detailed protocol is provided for differentiating human blood monocytes into anti-inflammatory macrophages, covering steps for cell isolation, polarization, and phenotypic characterization. Finally, this model is evaluated for its potential use in other pathologies, such as infections where MDMs heterogeneity closely reflects physiological conditions, offering a promising tool for research and therapeutic applications.