Cancer stem cells (CSCs) represent a critical subpopulation within tumors, endowed with self-renewal and differentiation capacities, and are implicated in tumor initiation, progression, metastasis, therapeutic resistance, and recurrence. This study sought to establish and validate a microfluidic device (MD) for the enrichment, functional assessment, and therapeutic evaluation of CSC populations derived from experimental models and primary tumor samples. Murine (LM38LP) and human (BPR6) breast cancer cell lines were cultured within MDs to promote sphere formation. CSC enrichment was confirmed through the expression analysis of pluripotency-associated genes (Oct4, Sox2, Nanog, and CD44) by means of quantitative PCR (qPCR) and immunofluorescence. Sphere number, size, and gene expression profiles were quantitatively assessed before (control) and after chemotherapeutic exposure. To validate the MD platform against a conventional scale, parallel experiments were performed in 12-well plates. To extend translational relevance, three primary canine tumor samples (solid thyroid carcinoma, simple tubular carcinoma, and reactive lymph node) were mechanically disaggregated and processed within MDs for CSC characterization. The MD platform enabled consistent CSC population enrichment, showing significant sphere growth modulation parameters and stemness marker expression following treatment. A notable reduction in both size and growth rate was observed in spheres treated with Doxorubicin or Paclitaxel after 8 days of culture, compared to controls. These findings are particularly significant, as this technique can be used to assess cell heterogeneity and the potential of cells to form tumors. The MD also supported immunofluorescence staining and allowed for real-time monitoring of individual cell growth. Sphere formation efficiency and CSC marker expression were demonstrated in primary veterinary tumor cultures, highlighting the device’s cross-species applicability. Microfluidic-based sphere assays represent a robust, reproducible, and scalable platform for the functional interrogation of CSC dynamics and therapeutic responses. This methodology holds great promise for advancing CSC-targeted therapies and supporting personalized oncology in both human and veterinary settings.