The study presents the development of a robust portable smartphone-based device for determining tobramycin in drugs and urine by a fluorescence-based method. As existing analytical methods for the determination of tobramycin often require complex equipment and time-consuming procedures, the proposed approach offers simplicity, portability and real-time monitoring capabilities. The spectrofluorimetric method is based on the reaction of tobramycin and fluorescamine in an alkaline medium. The measurement of analytical signals was enabled with a specially designed device integrating a 3D printed adapter, LEDs, a macro lens, an Arduino-based microcontroller, and asmartphone. The traditional spectrofluorimetry was used as a reference method. Preliminary and optimisation studies were conducted to select the most suitable experimental conditions (e.g. reagent concentration and reaction time) as well as instrumental parameters (e.g. selection of the appropriate RGB model component, ISO, and white balance). The performance of the method was validated, and then verified against conventional spectrofluorimetry, showing comparable accuracy and precision. The method implemented in a smartphone-based device showed a linear range of 0.10–1.00 mgL-1 with a detection limit of 0.03 mgL-1. Synthetic samples were tested to verify analytical usability, followed by analysis of pharmaceutical and urine samples. The method showed good precision (CV<3.4 %) and recovery values (92.3–114.1 %), consistent with spectrofluorimetric results (CV<5.9 %, recovery 92.3–119.8 %). Comparison with other methods highlighted the simplicity and efficiency of the developed approach. The research reveals that it is competitive with existing well-established methods. The proposed device offers reliable results with low reagent and sample consumption, making itsuitable for on-the-go analysis in non-laboratory conditions.