A comparative study of two types of silver nanoparticles was investigated. The effect of the surface chemistry of the studied silver nanoparticles (AgNPs) on their localized surface plasmon resonance was utilized for the quantitative determination of muscle relaxant tizanidine drug. The studied AgNPs are classified according to the type of stabilizing agent used in their synthesis into electrostatically and sterically stabilized AgNPs. The electrostatically-stabilized AgNPs (AA AgNPs) were prepared using ascorbic acid as both reducing and protective agents in alkaline medium. The sterically-stabilized AgNPs type (PEG-AA AgNPs) was prepared using ascorbic acid as a reducing agent and polyethylene glycol as a stabilizing agent at room temperature. The interaction of tizanidine with AgNPs was characterized using four different techniques including, transmission electron microscope, UV–visible spectrophotometric, surface enhanced Raman spectroscopic (SERS) and electroanalytical methods. SERS method was developed to study the relationship between the plasmon resonance and the enhanced power of Raman signal. The electrocatalytic behavior and the interfacial properties of AgNPs were studied using cyclic voltammetry and electrochemical impedance spectroscopy (EIS) on glassy carbon electrode modified with AgNPs. The quantitative determination of tizanidine in pharmaceutical and biological samples was successfully achieved by using AgNPs probe based on spectrophotometric methods. A linear response over the range 10–400 nmol L−1 was obtained. Validation procedures were carried out following International Conference on Harmonization (ICH) guidelines.