RNA molecules undergo a variety of modifications, including inosine modification, also called adenosine-to-inosine (A-to-I) RNA editing, which is prevalent across all domains of life. To unravel the roles of A-to-I RNA editing, it is essential to accurately quantify inosine in RNA at specific sites. Here, we developed a ligation-assisted qPCR (LA-PCR) method for the quantitative and site-specific analysis of A-to-I RNA editing. In LA-PCR, adenosine on an edit site pairs with thymidine. In contrast, inosine fails to pair with thymidine, disrupting the nick ligation of the two DNA probes located upstream and downstream from the editing site. The reduction in the liaged products can be quantified through subsequent qPCR, thus enabling the quantification of the A-to-I RNA editing level. The LA-PCR approach was successfully employed to detect and quantify the A-to-I RNA editing at position 2814 in Ino80dos RNA from mouse tissues. A notable elevation in A-to-I RNA editing levels was found across various tissues from sleep-deprived mice in comparison to control mice, suggesting a potential association between A-to-I RNA editing and sleep behavior. The proposed method facilitates the quantitative analysis of A-to-I RNA editing at specific sites, aiding in the elucidation of the functions and mechanisms of A-to-I RNA editing.