<p>The paper describes developing a gentamicin determination method using silver nanoparticles (AgNPs) and smartphone-based detection. The study included selecting conditions for AgNPs’ synthesis using gallic acid as a natural stabilising and reducing agent, selecting conditions for AgNPs aggregation induced by gentamicin, such as aggregation time and AgNPs concentration, choosing the measuring channel for smartphone-based detection, and testing the possibility of using different smartphones. Spectrophotometry was used as a reference method. Analytical parameters for spectrophotometric and smartphone-based detection methods, like the linear range: 30.00–90.00 µg dm⁻³, the limit of quantification: 30.00 µg dm⁻³_, the same for both methods, and precision (CV, n = 6) 6.5 and 2.6%, respectively, were determined. The selectivity of AgNPs under selected conditions against other antibiotics was also examined. The developed method was successfully applied to determine gentamicin in eye drop samples. Thanks to compact and accessible equipment and good analytical characteristics, it can become an alternative to other methods reported in the literature.</p> Graphical Abstract <p></p>