RNA modifications contribute to RNA and protein diversity in eukaryotes and lead to amino acid substitutions, deletions, and changes in gene expression levels. Several methods have developed to profile RNA modifications, however, a less laborious identification of inosine and pseudouridine modifications in the whole transcriptome is still not available. Herein, we address the first step of the above question by sequencing synthetic RNA constructs with inosine and pseudouridine modification using Oxford Nanopore Technology, which is a direct RNA sequencing platform for rapid detection of RNA modification in a relatively less labor-intensive manner. Our analysis of multiple nanopore parameters reveals mismatch error majorly distinguish unmodified versus modified nucleobase. Moreover, we have shown that acrylonitrile selective reactivity with inosine and pseudouridine generates a differential profile between the modified and treated construct. Our results offer a new methodology to harness selectively reactive chemical probe-based modification along with existing direct RNA sequencing methods to profile multiple RNA modifications on a single RNA. ### Competing Interest Statement The authors have declared no competing interest.