Epithelial-to-mesenchymal transition (EMT) is a proposed mechanism for initial metastatic invasion. Tumors are highly heterogeneous mixtures of tumor cells, and heterogeneous multicellular aggregates (MCAs) have emerged as in vitro surrogates for heterogeneous tumors. We custom-modified a commercial laser cutter to produce microwells by laser ablation of poly(dimethylsiloxane) (PDMS), which could generate MCAs with EMT phenotype. Tumor invasion is a dynamic process, but methods for longitudinal characterization of heterogeneous MCAs are lacking. We propose improvements to quantifying 1) MCA size using 2D maximum Feret diameter measured by automated image analysis; 2) Cell sub-population ratio determined from Imaris-mediated nuclei counting of cells with nuclear localized fluorescent proteins; and 3) Segmentation efficiency based on cell population overlap volume. Finally, we discuss directions for future longitudinal studies of EMT. Improvements to MCA production throughput and longitudinal characterization methods will improve studies of EMT with heterogeneous MCAs.