<p>The Analgesia Nociception Index (ANI) has been used to assess discomfort in anesthetized adults. The COMFORT Behavior Scale (CBS) is recommended for assessing discomfort in intubated and sedated children. The primary objective of the present study was to assess the validity and performance of the ANI as an indicator of discomfort in intubated, ventilated children in a pediatric intensive care unit (PICU). A prospective, non-interventional, single-center pilot study was conducted between June 1st, 2021, and November 31st, 2023. Intubated, sedated, prepubescent patients aged between 2 and 10&#xa0;years (for girls) or between 2 and 12&#xa0;years (for boys) were included. The instantaneous ANI (ANIi) and the mean ANI (ANIm) were recorded continuously during care procedures. Data were analyzed before (period (P)1), during (P2) and after (P3) care procedures. 50 patients were included; the median (interquartile range [IQR]) age was 7 [4; 9] years. The ANIi decreased significantly between P1 and P2 (median [IQR]: 59 [45; 80] vs. 33 [26; 42], respectively; p &lt; 0.0001) and increased significantly between P2 and P3 (33 [26; 42] vs. 51 [33; 72], respectively; p &lt; 0.0001). The CBS score increased significantly between P2 and P3 (median [IQR]: 10 [7; 13] vs. 12 [8; 15], respectively; p &lt; 0.0001). The ANIi was able to discriminate between over-analgosedation (defined as a CBS score &lt; 10) and normal analgosedation (AUROC = 0.694 during P1).The ANI might be a good candidate for assessing discomfort in intubated, prepubescent patients in the PICU.</p><p><i>Trial Registration</i>: NCT04913038</p>