Salvia miltiorrhiza is a medicinal plant highly appreciated by its content of tanshinones and salvianolic acids. Tanshinones are of particular relevance for their anti-oxidant, anti-tumoral and anti-inflammatory properties. Abiotic and biotic agents as silver nitrate and yeast extract have shown efficiently to stimulate tanshinone accumulation, but the underlying molecular mechanism remains essentially unknown. By using hairy roots as experimental material and the elicitors mentioned, were obtained up to 22 mg of tanshinones per gram of dry weight. Differential label-free quantitative proteomic analysis was applied to study the proteins involved in tanshinone biosynthesis. A total of 2650 proteins were identified in roots extracts, of which 893 showed statistically (p < 0.05) significant change in relative abundance compared to control roots, 251 proteins were upregulated and 642 downregulated. Among the upregulated proteins the predominant functional categories were metabolism (47%), stress defense (18%) and redox homeostasis (10%). Within the metabolism category, isoprenoid metabolism enzymes, cytochromes P450 and FAD-binding berberine proteins showed abundance profile linked to tanshinone concentration. The results presented here allowed to propose 5 new cytochromes P450 and 5 berberine enzymes as candidates to be involved into tanshinone biosynthesis, a novel finding that opens new avenues to improve tanshinone production through biotechnological approaches.