An increasing number of studies concerning solid cancers, including prostate cancer, are tending to demonstrate the predominant role of the interactions of tumor cells with their microenvironment, and underlining the relevance of therapeutic approaches co-targeting these two components. Artificial in vitro 3D culture models, such as spheroids, are therefore being designed to allow intercellular interactions between tumor cells and the matrix, under hypoxic conditions mimicking a microtumor. This project aims to develop and characterize a multicellular tumor spheroid (MCTS) model of human prostate cancer cells expressing PSMA, for in vitro drug screening. To this end, 1,000 cells/well were seeded in 100 µl of culture medium with 0.5% of methylcellulose in 96-well, non-adherent, V-shaped bottom plates. Bioluminescent imaging of the spheroids enabled the measurement of spheroid growth. From Day 7 of growth, immunofluorescence studies showed cellular proliferation (Ki-67), mainly located in the periphery of the spheroid section, associated with the formation of an apoptotic core (TUNEL). Scanning electron microscopy and fluorescent imaging (Lox-1 probe) showed the presence of an extracellular matrix and the installation of an oxygen gradient leading to the formation of a hypoxic area during growth. This hypoxia was correlated with increased VEGF excretion. Drug sensitivity was assessed on 2D and 3D cultures. The LNCaP-Luc spheroids are more resistant to docetaxel and TH-302, a hypoxia-activated prodrug, compared with cells grown in a monolayer. For docetaxel, this resistance increased with the spheroid growth stage, whereas the activity of TH-302 was potentiated by the hypoxic environment. In conclusion, the development of LNCaP-Luc cell MCTS provides a simple model mimicking a microtumor; it appears to be particularly well-suited to the validation of new therapeutic approaches targeting proliferation and the microenvironment.