Purpose: Through controlled comparative rabbit experiments and parallel patient studies, our purpose was to understand mechanisms underlying differences in efficacy and toxicity between intra-arterial chemotherapy (IAC) and intravenous chemotherapy (IVC). Methods: In rabbits, ocular tissue drug levels were measured following IAC and IVC. Retinal toxicity was assessed using electroretinography, fluorescein angiography, optical coherence tomography (OCT) and OCT angiography. Efficacy to eradicate retinoblastoma orthotopic xenografts was compared. In IAC and IVC patients, we measured blood carboplatin pharmacokinetics and compared efficacy and toxicity. Results: In rabbits receiving IAC, maximum carboplatin levels were 134 times greater in retina (P = 0.01) and 411 times greater in vitreous (P < 0.001), and total carboplatin (area under the curve) was 123 times greater in retina (P = 0.005) and 131 times greater in vitreous (P = 0.02) compared with IVC. Melphalan levels were 12 times greater (P = 0.003) in retina and 26 times greater in vitreous (P < 0.001) for IAC. Blood levels were not different. IAC melphalan (but not IV melphalan or IV carboplatin, etoposide, and vincristine) caused widespread apoptosis in retinoblastoma xenografts but no functional retinal toxicity or cytopenias. In patients, blood levels following IVC were greater (P < 0.001) but, when adjusted for treatment dose, were not statistically different. Per treatment cycle in patients, IVC caused higher rates of anemia (0.32 ± 0.29 vs. 0.01 ± 0.04; P = 0.0086), thrombocytopenia (0.5 ± 0.42 vs. 0.0 ± 0.0; P = 0.0042), and neutropenia (0.58 ± 0.3 vs. 0.31 ± 0.25; P = 0.032) but lower treatment success rates (P = 0.0017). Conclusions: The greater efficacy and lower systemic toxicity with IAC appear to be attributable to the greater ocular-to-systemic drug concentration ratio compared with IVC. Translational Relevance: Provides an overarching hypothesis for a mechanism of efficacy/toxicity to guide future drug development.