Microelectrode arrays (MEAs) are a valuable tool for non-invasive, high-spatial mapping of extracellular cardiac electrophysiology in both cardiomyocyte monolayers and cardiac tissue preparations. Neonatal rat ventricular myocytes (NRVMs) represent a useful model for assessing intercardiomyocyte conduction properties as they can be maintained in culture, spontaneously beating as a functional monolayer. Comprehensive methodological details for culturing NRVMs onto glass MEAs are required. The aim of this study was to optimise plating conditions for spontaneously beating NRVMs onto glass MEAs, and to maximise cardiomyocyte-electrode adherence and field potential recording capacity on adapted MEAs coated with the conductive polymer “Pedot”. Sprague-Dawley rats (1-2 days old) were euthanised by decapitation, hearts rapidly excised and enzymatically digested to isolate NRVMs. NRVMs were seeded at varying densities onto fibronectin-coated MEAs (60EcoMEA or 60PedotEcoMEA, MultiChannel Systems; electrode spacing 700μm, diameter 100μm, 8×8 matrix) and maintained in culture. To determine optimal culture conditions, 5-6 days post-isolation, field potentials were recorded and activation maps generated to compare plating methodologies (central cell droplet vs whole-MEA culture) and MEA surface properties (uncoated vs Pedot-coated) using a MEA2100 system at 37oC. Culture responsiveness to β-adrenergic stimulation (1μM isoproterenol) was also assessed. Comparisons between groups were performed with t-tests or one-way ANOVA, as appropriate. Differences were considered significant at P<0.05. Restricting culture of cardiomyocytes to the central electrode recording zone enhanced total signal detection capacity from 27.1% to 99.6%, allowing reliable measurement of rapid conduction velocities (mean conduction velocity: 23.8±1.1cm/s). Use of Pedot-coated MEAs significantly increased field potential amplitude (Pedot-coated vs uncoated: 2796±331.9μV vs 467.5±36.2μV; n=6, P<0.05). In optimised cultures, 1μM isoproterenol significantly increased the spontaneous beating rate (isoproterenol vs control: 144±17bpm vs 86±6bpm; n=3, P<0.05). This was associated with a small, but significant increase in conduction velocity (23.4±1.8cm/s vs 24.9±2.1cm/s; n=3, P<0.05), validating the culture’s responsiveness to positive chronotropic stimulation. This study highlights the importance of confining the plating of cell monolayers to the central recording matrix on the MEA, in addition to demonstrating the advantages of using Pedot-coated MEAs. Optimised cultures have been validated through demonstrating positive canonical responsiveness to β-adrenergic stimulation. This study provides baseline conditions and recording signal characterisation for use as reference in future investigations of conduction abnormality and arrhythmogenicity.