A sequential injection method for the determination of penicillamine (PA) was developed based on quenching the chemiluminescence generated by oxidation of luminol by hydrogen peroxide in presence of silver nanoparticles (AgNPs). The chemiluminescence (CL) of the reaction was found to greatly enhance in presence of AgNPs due to the increased catalyst surface area. The method was sensitive and found suitable for analysis of penicillamine in pharmaceutical preparations. Linear calibration curve is obtained in the range 0.2-1.0 mg mL-1 with a relative standard deviation less than 2%. A recovery percent of 102.3 ±0.2 was obtained with the tablets matrix indicating reasonable selectivity of the method for PA in tablets. The mechanism of quenching of the CL reaction was investigated by UV-Visible spectroscopy and transmission electron microscopy as well as by theoretical calculations using DFT-B3LYP method. The covalent attachment of PA to the AgNPs triggers aggregation of the particles thereby diminishing the surface significantly. The method was applied for the assay of PA in pharmaceutical preparations.