Monocytes are highly plastic cells and can be used as precursors for the generation of dendritic cell (DC)-based vaccines. Despite the capacity of DC vaccines to elicit antigen-specific T responses, their clinical benefit has been disappointing. Poor phenotypic stability, reduced migratory capacity, and the acquisition of tolerogenic features when injected into patients might explain their limited clinical efficacy. Consequently, tailoring the immunogenic potential of DCs by manipulating their differentiation offers the possibility of improving the efficacy of DC vaccines, especially in combination with immune checkpoint blockade therapies. We have reported that the simultaneous pharmacological inhibition of the mammalian target of rapamycin complex 1 and the peroxisome proliferator-activated receptor γ (PPARγ) in the presence of GM-CSF induces the differentiation of highly immunogenic human monocyte-derived dendritic cells (MoDCs). When compared to the traditional method to generate MoDCs in vitro, using GM-CSF and IL-4, these cells show higher phenotypic stability and stronger immunogenic profile. Here we provide a comprehensive protocol for the generation of these cells and evaluation of their immune functions in vitro.