Optical mapping techniques have been used to measure cardiac ventricular electrical excitation patterns, with high spatial and temporal resolutions, in whole hearts isolated from several species including mouse, rat, rabbit and guinea pig [1-5]. The shapes of the ventricular action potential (AP) are species-specific but they generally share a common feature that consists of a very fast upstroke and a much slower repolarisation. These AP kinetic differences pose a pragmatic challenge of how to reliably quantify dynamic features of membrane potential changes across each entire AP recorded simultaneously from several thousand pixels. In support of our studies of electrical excitation in isolated guinea pig hearts, we have developed algorithms and novel analysis procedures that improve the quantification of several dynamic parameters of cardiac APs. We have routinely recorded spatiotemporal propagation (1kHz, 128×128 pixels of ~230μm) of left ventricular APs from isolated guinea pig hearts for up to 60 seconds. Dynamic features of individual pixel APs are measured using a piece-wise digital filtering algorithm to: (i) securely separate the upstroke of the AP (ranging 8-15msec depending on biological setting) from the remainder of excitation; this enables accurate quantification of upstroke duration and speed. (ii) apply a suitable noise reduction filter on the plateau and repolarisation phase. This improves signal:noise, consistency of APs recorded between pixels, and facilitates accurate determination of AP durations (APDs) and repolarisation rates. Another notable improvement is made on the computation of the conduction velocity (CV) across the ventricle by enacting an algorithm that ensures the validity of the spread of data points surrounding each and every point of interest. These procedures facilitate the accurate quantification of entire AP spatiotemporal parameters including upstroke duration, APD and CV. The ventricular AP form and dynamics can vary considerably between commonly-used experimental models. For example, the APDs in mouse and rat (~40 – 50 msec [1,2]) are considerably shorter than that of guinea pigs (174 msec, 186 msec [3]) or rabbits (~155 – 176 msec [4,5]). The new method of analysis of AP dynamics derived from optical mapping signals described herein offers the opportunity to provide consistency to data measurements across different experimental models and, thereby, improve the assessment of cross-species differences in cardiac spatiotemporal excitation patterns.