Monitoring prognostic biomarkers is essential for evaluating cancer treatment efficacy in real time. Here, we present a rapid, proof-of-concept microfluidic assay for cancer biomarker quantification and functional therapy evaluation. The assay requires only 5 µL of sample, eliminates tedious sample manipulation, and uses commercial antibodies to track both biomarker concentration and functional activity. We demonstrated the successful detection of VEGF and EML4-ALK proteins in both lung cancer cell culture supernatant and serum-spiked samples, achieving a detection sensitivity 15 times greater than standard ELISA using the same antibody pairs for the respective antigens. Crucially, we showcase the platform’s distinct applicability to functional assays by monitoring dynamic phosphorylation changes in EML4-ALK following treatment with the tyrosine kinase inhibitor alectinib, successfully capturing a significant, rapid decrease in phosphorylation levels. At this preclinical stage, the platform demonstrates analytical and functional feasibility for highly sensitive and rapid biomarker monitoring, providing a foundation for future validation in patient-derived ALK-positive samples for therapeutic-response assessment.