Validation of ultraviolet (UV) reactors using Lagrangian Actinometry (LA) has been successfully applied to several large-scale UV reactors for drinking water disinfection. The primary advantage of LA is the ability of the method to yield a measurement of the UV-dose distribution of the reactor. An accurate measurement of the UV dose distribution can be used to provide an accurate prediction of reactor performance relative to any photochemical endpoint for which accurate UV dose-response behavior is known. In order to separate dyed microspheres (DMS) from background particles, it is desired to provide DMS with larger sizes and higher fluorescence intensity. Three types of microspheres with different diameters (nominally 5, 10 and 15 µm) and surface coating materials were tested. The 10 and 15 µm DMS were better separated from background particles in flow cytometry. In this research, action spectra of three different types of DMS were also established. The collimated MP source and bandpass optical filters used in these experiments allowed measurement of wavelength-dependent dose-response behavior for all three DMS types over the entire germicidal UV spectrum for UV doses in the range of 0-450 mJ/cm². Internal (microsphere) standards were applied for all samples to calibrate the measurements in flow cytometry. The action spectra of three different types of DMS were similar and wavelengths between 240 to 280 nm showed higher efficiency. A dose of 450 mJ/cm<sup>2>/sup> at the most sensitive wavelength gave mean FI increases of 50, 700 and 400 units for 5, 10 and 15 µm DMS, respectively. The 10 µm DMS provided the greatest sensitivity to UV radiation at all wavelengths. The higher sensitivity of DMS can improve not only the separation from background particles but also determination of dose distribution. Includes 6 references, table, figures.