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In vitro tumor spheroid models have been developed using microfluidic systems to generate 3-D hydrogel beads containing components of alginate and ECM protein, such as collagen, with high uniformity and throughput. During bead gelation, alginate acts as a fast gelling component helping to maintain the spherical shape of beads and to prevent adjacent or underlying beads from coalescing when working with the slower gelling temperature and pH-sensitivity of collagen components. There are also well-known limitations in using microfluidic systems when working with temperature-sensitive components of collagen type I, and it is determined that to produce uniform hydrogel droplets through a microfluidic system, the mixtures must be homogeneous. However, the issue of collagen’s sensitivity to temperature causes concern for chunks of collagen gel inside of the mixture before bead encapsulation; therefore causing the mixture to become non-uniform and risking chip clogging. In order to overcome this limitation, previous approaches have used a cooling system during bead encapsulation while tumor cells were also present in the mixture, but this procedure assisted in postponing collagen gelation prior to bead production and potentially contributing to a delay in cell proliferation. Here a novel yet simple method is developed to prepare homogeneous pre-bead-encapsulation-mixtures containing collagen through ultrasonication, while extending cell viability and proliferation. This method allows the cultivation of homogenous TS cultures with high uniformity and compact structure, and not only maintains cell viability but also stimulates the proliferation of cells in alginate/collagen hydrogel bead cultures. Depending on the sonication parameters, time and temperature, gelation of collagen is controlled by small sized fibrils to thick fibers. Human-source-Michigan-Cancer-Foundation-7 (MCF-7) cells isolated from a breast cancer cell line are successfully incorporated into alginate/collagen mixtures, followed by sonication, and then bead production. After bead gelation, the encapsulated MCF-7 cells remained viable and proliferated to form uniform TSs when the beads contained alginate and collagen. Results indicate that ultrasound treatment provides a powerful technique to change the structure of collagen from fiber to fibril, and to disperse collagen fibers in the mixture homogeneously for an application to generate uniform hydrogel beads and spheroids while not disturbing cell proliferation.