Integrating serum pharmacochemistry, network pharmacology, and molecular docking with in vivo experimental validation to investigate the therapeutic effects and mechanism of the Rehmanniae Radix-Salviae Miltiorrhizae Radix (RRSM) against diabetic retinopathy (DR). A DR rat model was established to evaluate the anti-DR effects of RRSM in vivo. Serum pharmacochemistry combined with UPLC-Q-Orbitrap HRMS analysis was employed to identify the prototype components of RRSM absorbed into systemic circulation. Network pharmacology was then applied to predict the potential mechanisms of RRSM against DR, followed by molecular docking and in vivo experiments to validate the network pharmacology predictions. The results demonstrated that RRSM exhibited therapeutic effects on DR rats. Serum pharmacochemical analysis identified 22 prototype components of RRSM in the blood. Network pharmacology revealed 548 RRSM-related targets and 2 646 DR-related targets, with 239 overlapping targets. Protein-protein interaction  network analysis screened 35 core targets. KEGG pathway enrichment indicated that RRSM modulated multiple DR-associated pathways, with the advanced glycosylation end products/receptor of AGEs (AGEs/RAGE) and phosphoinositide 3-kinase/protein kinase B (PI3K/AKT) pathways showing the highest enrichment. Molecular docking confirmed strong binding affinity between RRSM's key active components and critical nodes (RAGE/PI3K/AKT). In vivo experiments showed that, compared to the model group, the RRSM group exhibited significantly elevated protein expression of RAGE, PI3K, and AKT (P < 0.05), alongside markedly reduced levels of nuclear factor κB(NF-κB), vascular endothelial growth factor (VEGF), tumour necrosis factor-α (TNF-α), and interleukin-1β (IL-1β) (P < 0.05), with superior efficacy to single-herb treatments. This study preliminarily elucidates that RRSM alleviates DR by regulating the RAGE/PI3K/AKT pathway to exert anti-inflammatory and anti-angiogenic effects, providing a scientific basis for exploring herbal compatibility in DR treatment.