Considerable advances in imaging-based biomarker discovery paved ways for more accurate diagnosis of complex diseases such as cancer. Recently, AIMP2-DX2, an alternatively spliced form of AIMP2, was unveiled as a new cancer-inducing factor. Understanding of AIMP2-DX2 and its role in various diseases at the RNA level will better our understanding to exploit its expression level as a biomarker. However, accurately detecting its RNA expression in vivo and in the clinic remains challenging considering some associated alternative splicing alterations and the difficulty to distinguish them from its normal RNA form. This limits the potential utility of AIMP2-DX2 as a biomarker. Here, we proposed a novel RNA-in situ hybridization (RNA-ISH) platform as a detection method for measuring the expression of AIMP2-DX2, and validated its accuracy and potential to be used as an image-based biomarker. We generated two types of RNA-ISH probes designed for binding to specific sites of AIMP2 mRNA, exon 2 or not exon 2, and confirmed the binding specificity by staining various cancer cell lines with different fluorophores. We also consulted published NGS datasets to corroborate AIMP2 RNA expression levels with our RNA-ISH results. Further research is on-going to improve the probes and adapt the protocol to accommodate other complex tissue samples. This unique detection method could improve the understanding of the function of AIMP2-DX2 and its implications in the pathogenesis of various cancers.