Due to widespread usage of antibiotics, the possibility of surface and groundwater contamination with such pollutants has significantly increased over the years as such antibiotics have been widely reported in water bodies. This study reports on the elimination of toxic antibiotic Penicillin G (PG) from wastewaters sources via the sonochemical process in the presence of hydrogen peroxide (H2O2) as well as to identify the degradation by-products. The effect of initial pH, pollutant dosage and presence of hydrogen peroxide on the degradability was examined. Response Surface Methodology (RSM) was used to model the sonochemical process with high correlation of (R2= 0.9784) and predictions agreed well with the experimental results. Box-Behnken Design (BBD) and RSM equations were used to determine the optimal values of experimental conditions for the PG degradation at the optimum conditions: PG dose of 20 mgL−1, pH 3.4 and hydrogen peroxide (H2O2) concentration of 600 mgL−1. Under the optimized conditions, removal efficiency reached 97% and by-products formed during sonochemical degradation were identified via QuEChERS method (Quick, Easy, Cheap, Effective, Rugged, and Safe), while the pre-concentration by evaporation was followed by the direct infusion-high resolution mass spectrometry technique (DI-HRMS). This is the first study to report a detailed investigation on PG degradation products under the sonochemical treatment combined with QuEChERS method to DI-HRMS techniques. Such advanced methods allowed us to determine the analyte substance found in water at low concentrations (ng L−1). The seven by-products have been detected where two possible degradation pathways were proposed with a resulting total elimination of PG.