Recent research indicates that cancer stem-like cells (CSCs) are the key subpopulation for tumor relapse and metastasis. Due to cancer plasticity in surface antigen and enzymatic activity markers, functional tumorsphere assays are promising alternatives for CSC identification. To reliably quantify rare CSCs (1-5%), thousands of single-cell suspension cultures are needed. While microfluidics is a powerful tool in handling single cells, previous works provided a limited throughput and lack in automatic data analysis required for high-throughput assays. In this work, we present a highthroughput single-cell-derived tumor sphere assay chip, facilitating the tracking of up to ∼10,000 cells in each chip with ∼76.5% capture rate. The presented cell capture scheme guarantees sampling of a representative population from the bulk cells. Combined with F108 coating on PDMS, a suspension culture environment was created for high-throughput tumorsphere assays. To analyze thousands of single-cells, a highly adaptable analysis program was developed for cell/sphere counting and size measurement for automation in CSC heterogeneity studies.