Antimicrobial susceptibility testing plays a pivotal role in the discovery of new antibi-otics. However, the development of simple, sensitive, and rapid assessment approaches remains challenging. Herein, we report an activated alkyne- based cascade signal ampli-fication strategy for ultrafast and high-throughput antibiotic screening. First of all, a novel water-soluble aggregation-induced emission (AIE) luminogen is synthesized, which contains an activated alkyne group to enable fluorescence turn -on and metal -free click bioconjugation under physiological conditions. Taking advantage of the in -house established method for bacterial lysis, a number of clickable biological substances (i.e., bacterial solutes and debris) are released from the bacterial bodies, which remarkably increases the quantity of analytes. By means of the activated alkyne-mediated turn -on click bioconjugation, the system fluorescence signal is significantly amplified due to the increased labeling sites as well as the AIE effect. Such a cascade signal amplification strategy efficiently improves the detection sensitivity and thus enables ultrafast antimi-crobial susceptibility assessment. By integration with a microplate reader, this approach is further applied to high-throughput antibiotic screening.