In this work, a new spectrophotometric ultrasonicated method was applied for the determination of diaverdine HCl (DIA). The process was based on a controlled preparation of silver nanoparticles stabilized with sodium dodecyl sulphate (SDS-AgNPs), and then the obtained solution was subjected for a deinite time to ultrasonication, which in turn increased the absorbance of surface plasmon resonance (SPR), at λ max = 395 nm, by 50%. The analyte (DIA) was added to freshly sonicated SDS-AgNPs, and the DIA concentration was monitored with respect to the decreasing SPR absorption spectra. The obtained results and the mechanism of silver nanoparticle aggregation due to DIA addition were conirmed using the data obtained from FT-IR and TEM measurements. A spectrophotometric determination of DIA was valid with a linear range of 0.1–1.5 μg  mL with R 2 and relative standard deviation of 0.992 and 1.72%, respectively. The developed method was successfully applied for the monitoring of DIA in spiked bovine milk samples and a veterinary drug sample. −1