Proteins are widely used as drug targets, enzyme substrates, and biomarkers for numerous diseases. The emerging demand for proteins quantitation has been increasing in multiple fields. Currently, there is still a big gap for high-throughput protein quantitation at intact protein level using label-free method. Here we choose ribonuclease B (RNB) as a model, which is the substrate for human endo-β-N-acetylglucosaminidase (hENGase), a promising drug target for the treatment of N-Glycanase deficiency. Intact proteinlevel multiple reaction monitoring (MRM) methods were initally developed and optimized to quantify RNB and deglycosylated RNB (RNB-deg), with the S/N ratio improved by nearly 20-fold compared to the traditional full MS scan methods. To further increase the throughput making it possible for hENGase inhibitors screen, the protein MRM methods were introduced to the RapidFire-MS/MS system, achieving at least 12-fold throughput improvement. This assay was further optimized into 384-well plate format for compound screening with S/B ratio >37-fold and Z' factor >0.7 that is suitable for high-throughput screening of compound collections with a speed of 2 h per 384-well plate and an ability to screen over 3000 compounds per day at a single concentration dose. This 384-well plate based automated SPE-MS/MS assay is efficient and robust for compound screening and the assay format has a wide applicability to protein targets for other disease models.