Endometriosis is a painful gynecological condition characterized by ectopic growth of endometrial cells. Little is known about its pathogenesis, which is partially due to a lack of suitable experimental models. Here, we use endometrial stromal (St-T1b), primary endometriotic stromal, epithelial endometriotic (12Z) and co-culture (1:1 St-T1b:12Z) spheroids to mimic the architecture of endometrium, and either collagen I or Matrigel to model ectopic locations. Stromal spheroids, but not single cells, assumed coordinated directional migration followed by matrix remodeling of collagen I on day 5 or 7, resembling ectopic lesions. While generally a higher area fold increase of spheroids occurred on collagen I compared to Matrigel, directional migration was not observed in co-culture or in 12Z cells. The fold increase in area on collagen I was significantly reduced by MMP inhibition in stromal but not 12Z cells. Inhibiting ROCK signalling responsible for actomyosin contraction increased the fold increase of area and metabolic activity compared to untreated controls on Matrigel. The number of protrusions emanating from 12Z spheroids on Matrigel was decreased by microRNA miR-200b and increased by miR-145. This study demonstrates that spheroid assay is a promising pre-clinical tool that can be used to evaluate small molecule drugs and microRNA-based therapeutics for endometriosis.