ARS-interacting multifunctional protein 2 (AIMP2) is a non-enzymatic component required for the assembly of the multi-tRNA synthetase complex. Yet, it also serves as a multifunctional tumor suppressor. While exon 2 skipping variant of AIMP2 (AIMP2-DX2) is associated with carcinogenesis, its potential as a cancer biomarker remains uninvestigated. Here, we combine RNA in situ hybridization (RNA-ISH), quantitative image-analysis, and RNA-sequencing to develop clinically applicable detection tools that can quantitate the expression ratio of AIMP2-DX2 to AIMP2. We apply our technique to analyze AIMP2-DX2 expression levels in both cell lines and patients’ samples. Our image quantification results show excellent concordance with pathologist’s reading and tissue-matched RNA-sequencing. We further employ our tool and find that Alu-binding proteins, especially ADAR and PKR, can regulate AIMP2-DX2 expression. Collectively, we provide the development and application of RNA-ISH image-analysis tools for quantitative analysis of AIMP2-DX2 and subclassification cancer patients based on AIMP2-DX2 expression.