Abstract Measurement of drug distribution in the inner ear has important roles in the design of local delivery methods, such as direct, intracochlear delivery, and in assessment of emerging drug candidates in preclinical animal models. Sampling methods have been used in the past to measure drug concentrations in the cochlear fluids, but these methods provide no direct information about drug distribution in the cochlear tissues. In this work, we evaluated four fluorescent markers that simulate drug distribution in the organ of Corti after intracochlear delivery to the cochlea's scala tympani compartment. Our hypothesis is that ultimately, a cocktail comprising several fluorescent drug surrogates or fluorescently-tagged drugs, each with differing distribution, spreading, and clearance behavior, can be used to evaluate both transient and cumulative drug distributions associated with different delivery techniques. In this study, FITC-dextran, Qtracker™ 655, gentamicin Texas-Red, and FM 1–43 FX were each evaluated as candidate markers by direct intracochlear infusion into guinea-pig cochleae. Distribution of the markers was measured using fluorescence confocal microscopy imaging of cochlear whole mount dissections from animals sacrificed 3 h after the tracer-infusion. For all four tracers, strong fluorescence was observed in the tissue sections near the base, but only Qtracker™-655, gentamicin Texas-Red (GTTR) and FM 1–43 FX exhibited any specificity in labelling of the sensory hair cells. Therefore, these substances represent leading candidates for the quantification drug distribution achieved by different delivery approaches to the scala tympani. Graphical abstract Cannula insertion point within Scala tympani. Unlabelled Image [ABSTRACT FROM AUTHOR]