Study of cellular senescence is critical in aging research and anti-senescence therapy drug development. Current methods for the evaluation of the widely accepted cellular senescence marker senescence-associated beta galactosidase (SA-β-gal) activity assay rely on bright-field imaging, which is non-quantitative and tedious to perform. We have developed an effective and reproducible multiplex high-content analysis system for high-throughput screen and evaluation of senescence modulators. The IC(50) or EC(50) of the senescence modulators on fibroblasts were determined, which are essential for drug development and have not been able done before. In a single high-throughput screen, all three types of senomorphic, senolytic, and seno-inducing agents can be evaluated effectively. Using our system, we have identified new molecular entities that modulate MMC-induced senescence in fibroblasts through potential new cellular targets.