This work provides a new insight on fundamental principles of the interaction mechanism between two forms of tetracaine – a potent local anesthetic – both in neutral (TC) and ionized (TC+) states, with beta- (β-CD) and hydroxypropyl-beta-cyclodextrin (HP-β-CD), and how such interactions affect the transport of tetracaine, at different concentrations, across a model membrane. The kinetics and mechanism of TC release from HPMC gels is also evaluated giving an insight on the role of cyclodextrin on the tetracaine transport. HPLC, fluorescence and NMR spectroscopies provided solid physicochemical knowledge of these systems and in vitro studies were performed to obtain relevant data on the transport and mechanism parameters. HPLC and fluorescence spectroscopy data revealed that tetracaine interacts with both cyclodextrins on a 1:1 stoichiometry but it is observed that neutral tetracaine forms more stables complexes (ca. 1050 M−1 for both cyclodextrins) than in its ionized form (628 and 337 M−1 for β-CD and HP-β-CD respectively). Despite of that, no host–guest interactions take place as seen by ROESY. This study clearly demonstrates that both forms of tetracaine are successfully released from the formulations at a controlled rate, following a Super-Case transport mechanism and the transport of tetracaine can be tuned by using cyclodextrins.