AbstractINTRODUCTIONJanus kinase (JAK) inhibitors were recently identified as promising drug candidates for repurposing in Alzheimer's disease (AD) due to their capacity to suppress inflammation via modulation of JAK/STAT signaling pathways. Besides interaction with primary therapeutic targets, JAK inhibitor drugs frequently interact with unintended, often unknown, biological off‐targets, leading to associated effects. Nevertheless, the relevance of JAK inhibitors’ off‐target interactions in the context of AD remains unclear.METHODSPutative off‐targets of baricitinib and tofacitinib were predicted using a machine learning (ML) approach. After screening scientific literature, off‐targets were filtered based on their relevance to AD. Targets that had not been previously identified as off‐targets of baricitinib or tofacitinib were subsequently tested using biochemical or cell‐based assays. From those, active concentrations were compared to bioavailable concentrations in the brain predicted by physiologically based pharmacokinetic (PBPK) modeling.RESULTSWith the aid of ML and in vitro activity assays, we identified two enzymes previously unknown to be inhibited by baricitinib, namely casein kinase 2 subunit alpha 2 (CK2‐α2) and dual leucine zipper kinase (MAP3K12), both with binding constant (K_d) values of 5.8 μM. Predicted maximum concentrations of baricitinib in brain tissue using PBPK modeling range from 1.3 to 23 nM, which is two to three orders of magnitude below the corresponding binding constant.CONCLUSIONIn this study, we extended the list of baricitinib off‐targets that are potentially relevant for AD progression and predicted drug distribution in the brain. The results suggest a low likelihood of successful repurposing in AD due to low brain permeability, even at the maximum recommended daily dose. While additional research is needed to evaluate the potential impact of the off‐target interaction on AD, the combined approach of ML‐based target prediction, in vitro confirmation, and PBPK modeling may help prioritize drugs with a high likelihood of being effectively repurposed for AD.Highlights This study explored JAK inhibitors' off‐targets in AD using a multidisciplinary approach. We combined machine learning, in vitro tests, and PBPK modelling to predict and validate new off‐target interactions of tofacitinib and baricitinib in AD. Previously unknown inhibition of two enzymes (CK2‐a2 and MAP3K12) by baricitinib were confirmed using in vitro experiments. Our PBPK model indicates that baricitinib low brain permeability limits AD repurposing. The proposed multidisciplinary approach optimizes drug repurposing efforts in AD research.