Migration ability is an important feature of tumor cells. There are several approaches to analyze the dynamics of cancer cell migration in vitro. One of the most perspective and closer to the in vivo conditions is the model of initiation of the cell migration from 3D multicellular spheroids onto growth surface. Aim. Optimization of the model for adequate quantitative characteristics of the tumor cell locomotion during several days. Methods. 2D and 3D MCF-7 cell culture, immunofluorescence analysis, and image analysis using computer software Fiji. Results. Unification of spheroid size allowed avoiding a significant data deviation. The obtained spheroids spread completely for 3 days. The highest migration ratio was observed at the 2nd day. The proliferation level at each of 3-day experiment was the same and did not exceed 3%. The validity of the model was tested after migration inhibition by rapamycin (mTOR signaling inhibitor). Additionally, this model was successfully applied to immunofluorescence analysis, namely investigation of p85S6K1 subcellular localization in moving MCF-7 cells. Conclusions. Double filtration of multicellular spheroids allowed unification of their size, which promotes an adequate interpretation of the migration assay. This model enabled the study of tumor cells migration dynamics and can be further used for the development of anticancer drug.