This work describes the construction and evaluation of a fluorometer for use in paper analytical devices, using a smartphone to operate the instrument and to perform real-time image-based detection. In this approach, a circular PAD containing twenty analytical plates is rotated at 18° increments under a UV LED source, providing a sequential irradiation of plates and the detection of the luminescence with a lab-made application, capable of automatically identifying the analytical zones and collecting the RGB intensities from the selected pixels. As a proof of concept, the fluorometer performance was evaluated for the determination of quinine in beverages and riboflavin (B2 vitamin) in supplements. Quinine, which is less photoreactive, provided steady-state signals, while riboflavin, which rapidly degrades under UV photons, presented transient responses for RGB detection. For both analytes, linear calibration ranges (R² > 0.99) were observed from 2.0 mg L⁻¹ to 10.0 mg L⁻¹ with limits of detection estimated at approximately 1.0 mg L⁻¹. Nevertheless, it was demonstrated that successive additions of standard solutions to a single analytical plate of PAD could enhance the signal-to-noise ratios for less concentrated samples, acting as a pre-concentration step. In addition, suitable deviations for the signals (ca. 4.0%) and the absence of systematic errors for most samples (9 out of 11), when compared with a reference method at 95% confidence level, indicates that the proposed strategy is precise and accurate enough to be used as analytical tool for fluorescence detection in PAD.