A method and a system for rapid kinetic fluorescence measurement in high-throughput screening with high time-resolution includes providing a microtiter plate having a transparent base and multiple assay wells, a dispensing system arranged above the microtiter plate for simultaneously adding liquid into multiple assay wells, an illumination system beneath the microtiter plate for illuminating the multiple assay wells simultaneously through the transparent base, a detection system for detecting electromagnetic radiation from the multiple assay wells simultaneously through the transparent base, adding 0.3-300 μl of liquid per assay well from the dispensing system into the multiple assay wells, illuminating the multiple assay wells simultaneously through the transparent base using the illumination system before or from the time point of the start of the addition, detecting the electromagnetic radiation from the multiple assay wells simultaneously at a time interval of 1-1000 ms between individual measurement points.