Extracellular signal-regulated kinases 1 and 2 (ERK1/2) phosphorylate a variety of substrates important for survival and proliferation, and their activity is frequently deregulated in tumors. ERK pathway inhibitors have shown clinical efficacy as anti-cancer drugs, but most patients eventually relapse due to reactivation of the pathway. One factor limiting the efficacy of current therapeutics is the difficulty in reaching clinically effective inhibition of the ERK pathway in the absence of on-target toxicities. Here, we describe an assay suitable for high throughput screening to discover substrate selective ERK1/2 inhibitors, which may have a larger therapeutic window than conventional inhibitors. Specifically, we aim to target a substrate-binding pocket within the ERK1/2 catalytic domain outside of the catalytic cleft. The assay uses an AlphaScreen format to detect phosphorylation of a high-efficiency substrate harboring an essential docking site motif. Pilot screening established that the assay is suitably robust for high-throughput screening. Importantly, the assay can be conducted at high ATP concentrations, which we show reduces the discovery of conventional ATP-competitive inhibitors. These studies provide the basis for high-throughput screens to discover new classes of non-conventional ERK1/2 inhibitors.