<p>Most antibiotics have been demonstrated to be low biodegradable in biological treatment systems, thus advanced oxidation processes, particularly solar photocatalytic oxidation also known as a green process, have gained an essential attraction for their effective removal from effluents. However, so far there has been a very limited number of studies on ampicillin (AMP) removal that focused on one initial concentration of AMP and fixed photon energy. Therefore, this study was designed to investigate the effect of varying pH and incident photon fluxes on heterogeneous photocatalytic AMP removal at two AMP initial concentrations (50 and 100 mg L<sup>−1</sup>) using 0.5 g L<sup>−1</sup> TiO<sub>2</sub> dose under UV-A (315–400 nm wavelengths) irradiation. Photocatalytic experiments were run in a vessel with 200-mL effective sample volume. Process efficiency was monitored by degradation (UV–vis, LC-MS/MS), mineralization (TOC), and acute toxicity to <i>Daphnia magna</i> during 24–48 h exposure times to evaluate possible toxic effect of oxidation by-products. Toxicity results and TOC removal (30–60 min) indicated simultaneous mineralization and degradation of both initial AMP concentrations. Lower ratio of TiO<sub>2</sub>/AMP, as one of the key factors affecting the oxidation efficiency, promoted increasing formation of by-products that interacted with surface chemistry on TiO<sub>2</sub> nanoparticles leading to a process efficiency decrease at 100 mg L<sup>−1</sup> AMP initial concentration. When photon energy was increased, it enhanced the removal of AMP. A preliminary cost evaluation showed that a 3.62 mW cm<sup>−2</sup> energy flux was satisfying to obtain over 50% of TOC removal and a nearly complete detoxification of 50 mg L<sup>−1</sup> AMP.</p