Evidence-based therapeutic decision making is critical for precision cancer therapy. In our previous research, we developed an in vitro droplet microfluidic chip assay for cytotoxicity screening of chemical drugs against cancer cells. This one cell type model greatly restricted its application to chemotherapy and targeted therapy. Here we presented an improved assay applied with high content imaging to setup a two cell type model for cytotoxicity assessment of anti-CD19 chimeric antigen receptor T (CAR-T) cells against Nalm6 cancer cells expressing green fluorescent protein (GFP). This assay provides direct evidence for cytotoxic activity and facilitates mechanistic study by phenotype analysis.