Background <p>Entrectinib, a multi-target tyrosine kinase inhibitor (TKI) against TRK, ROS1, and ALK, is clinically approved for genetically-defined solid tumors. The prevalence of drug–drug interactions (DDIs) with increased use indicates the need for further research.</p> Objective <p>This present study aimed to screen 32 cardiovascular drugs for inhibitory effects on entrectinib metabolism and to elucidate the pharmacokinetic interaction with nicardipine.</p> Methods <p>We utilized rat liver microsomes to screen cardiovascular drugs and identify potent inhibitors. The inhibition kinetics of nicardipine were determined in vitro, and molecular docking to the primary entrectinib-metabolizing cytochrome P450 isoform was performed to investigate a potential mechanism for the observed interaction. Furthermore, in vivo pharmacokinetic studies were conducted in rats to evaluate the impact of nicardipine on entrectinib exposure.</p> Results <p>In rat liver microsomes, nicardipine was identified as a moderately potent inhibitor with a half-maximal inhibitory concentration (IC₅₀) of 1.43&#xa0;µM, inhibiting entrectinib metabolism through dual competitive and noncompetitive mechanisms (inhibition constants: <i>K</i>_i&#xa0;=&#xa0;1.28&#xa0;µM; α<i>K</i>_i&#xa0;=&#xa0;1.92&#xa0;µM). In vivo studies showed that nicardipine significantly increased the area under the curve (AUC) and maximum plasma concentration (<i>C</i>_max) of entrectinib by ~&#xa0;1.5-fold, while reducing clearance (CLz/<i>F</i>) and volume of distribution (Vz/<i>F</i>) (<i>p</i>&#xa0;&lt;&#xa0;0.05).</p> Conclusions <p>These findings in rat models and in silico docking indicate that nicardipine increases entrectinib exposure. While these results underscore the risk of significant DDIs, further clinical studies in humans are required to confirm this interaction and determine if entrectinib dose adjustments are necessary to mitigate adverse events.</p>