<p>Nasal drug delivery offers several advantages, such as rapid onset and avoidance of hepatic first-pass metabolism, making it a promising option for delivering drugs both locally and systemically. In the early phases of pharmaceutical development, predictive tools for nasal absorption are critical for selecting suitable candidates. This study evaluates the utility of RPMI 2650 cells as an <i>in vitro</i> model for predicting nasal drug absorption. First, we confirmed the reproducibility of membrane permeability across two air–liquid interface culture conditions (shifted on days 1 and 3), demonstrating consistent permeability profiles and agreement with previously reported <i>P</i>_app values. We investigated the correlation between the permeability of RPMI 2650 cells and both clinical nasal bioavailability (nasal BA) and the estimated human nasal fraction absorbed (nasal Fa), revealing good correlations in both cases. To our knowledge, this is the first study to demonstrate a significant correlation between <i>in vitro</i> permeability and estimated human nasal Fa levels. Although some compounds deviated from the correlation curve, these findings indicate that RPMI 2650 cells are useful <i>in vitro</i> tools for predicting nasal drug absorption during the early phases of pharmaceutical development.</p> Graphical Abstract <p></p>