Emopamil binding protein (EBP) is a transmembrane isomerase that catalyzes the conversion of 8,9-unsaturated sterols to 7,8-unsaturated sterols in the cholesterol biosynthesis pathway. Accumulation of 8,9-unsaturated sterols promotes oligodendrocyte progenitor cell (OPC) differentiation into new myelinating oligodendrocytes, making EBP inhibition a promising therapeutic strategy for neurodegenerative diseases such as multiple sclerosis. Structure-activity relationship (SAR)-driving assays are critical for advancing drug candidates through preclinical drug discovery. However, EBP is a challenging target due to low production yield and loss of enzymatic activity during the purification process. In addition, assay throughput is limited by chromatographic separation of EBP isomers. Here, we describe a robust functional assay to support SAR of EBP small molecule inhibitors. Immuno-mobilization of the active form of EBP, overexpressed in Expi293F cells, was coupled with liquid chromatography-atmospheric pressure chemical ionization multiple reaction monitoring MS (LC-APCI MRM MS) to quantify the potency of EBP inhibitors. This approach significantly enhanced EBP enzymatic activity at nanomolar concentrations compared to that of purified recombinant EBP (recEBP). Furthermore, automating sterol extraction and integrating a dual-arm high-speed LC autosampler further improved assay throughput. This approach showed excellent reproducibility and robustness suitable for SAR development. This was evidenced by high-quality individual concentration-response curves (CRCs) with confidence limit ratios (CLRs) below 5; a normal distribution of reference EBP inhibitor pIC50 values with a two-standard deviation (2-SD) of 0.61 based on 72 independent measurements; and consistent IC50 values for over 20 active EBP inhibitors spanning a broad potency range across two independent assay runs, with a mean pIC50 difference between runs of -0.05 and a 2-SD interval of 0.24.