Madder (Rubia tinctorum L.) is a perennial plant that its roots and rhizomes have rich anthraquinone (AQ) derivatives including alizarin and purpurin. This study was carried out to determine the effects of salicylic acid (SA) and L-phenylalanine (L-Phe) applications on the root growth parameters and secondary metabolite accumulation in madder adventitious roots derived from internode parts without needing to be collected from nature. For this aim, two different L-Phe (50 and 100 µM) and SA (20 and 40 µM) were added separately and together to the liquid Murashige and Skoog (MS) medium cultured adventitious roots for 7 days. Then roots were evaluated in terms of fresh root weight, root growth index, dry root weight, and contents of total AQ, alizarin, purpurin, total phenolic contents (TPC), and some important phenolic compounds. According to the results, L-Phe stimulated the root growth of madder while the effects of SA on root growth parameters varied depending upon its concentrations. L-Phe had no significant influence on the total AQ, alizarin, and purpurin. Conversely, SA increased the AQs, and 20 µM SA was the most suitable application providing the greatest total AQ, alizarin, and purpurin. TPC and individual phenolic compounds changed according to the applications. Not only L-Phe but also SA had positive effects on the phenolic accumulation in adventitious roots. It was determined that the combinations of 40 µM SA and 100 µM L-Phe were the most effective applications in terms of phenolic accumulation.