Three-dimensional (3D) cell culture models provide physiologically relevant systems that mimic the native endometrial environment better than 2D models and offer reliable platforms to study embryo implantation and maternal–embryo interactions. One widely used 3D culture model is the generation of spheroids. However, standardized and reproducible methods for generating uniform spheroids from trophoblast and endometrial stromal cells are limited. In this study, we established and validated a robust protocol for spheroid formation using human trophoblast (HTR8/SVneo, JEG3) and endometrial stromal (St-T1b, tHESC) cell lines. The protocol was further extended to generate spheroids from decidualized tHESC, representing a novel approach that closely reflects the receptive endometrial environment. Key parameters, including cell concentration and methyl cellulose supplementation, were optimized to produce compact and homogeneous spheroids. Spheroid formation was monitored at defined intervals (0, 8, 24, 32, and 48 h), and decidualized spheroids were assessed up to 72 h. Long-term cryopreservation over 11 months demonstrated high post-thaw viability across all spheroid types, as confirmed by Calcein-AM staining. This standardized workflow provides a reliable 3D model incorporating hormonally primed stromal cells and offers a practical platform to investigate the mechanisms underlying normal and trophoblast invasion in vitro.