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The non-invasive technique of optical mapping by using voltage-sensitive fluorescent dyes (VSFD), especially near-infrared (NIR) dyes. enables precise evaluation of electrical excitation propagation in the myocardium. However, in spite of considerable advantages when comparing with other clinical methods, OM is limited due to complicated data interpretation, fluorescent dye toxicity, and phototoxic effect, therefore, OM is used only in experimental studies. In this study we investigated the toxicity and phototoxicity of the novel NIR VSFD di 4 ANBDQBS, and their influence on the optical action potential (OAP) upstroke. The OAP upstroke provides information about electrical exitation propagation in cardiac tissue. The experiments were performed using the isolated cardiac cells of the rabbit heart, the right ventricle wall of the guinea pig heart, and the Langerdorf-perfused rabbit heart model. The results revealed that concentrations of 10–50 µM of di-4-ANBDQBS dye did not cause toxicity effects, and only 100 μM was determined as a toxic threshold which causes cardiac cell death. Also, we did not observe the phototoxic effect of the investigated dye in preparations of guinea pig ventricles. Registered temporary AP changes were just surface temperature variation that was caused by the light source. These temporary changes were not related with the phototoxic effect of the dye. Obtained transmural recordings of electrical APs via multiple cell layers in various depths of the LV wall enabled the calculation of averaged transmural-AP (TAP) and depth weighted DWTAP, which allowed evaluation of the OAP upstroke. The direction of excitation wave was simulated by artificial pacing from various cardiac surfaces. Simultaneous registration of OAP (by using di 4 ANBDQBS) and DWTAP (with microelectrodes) enabled to split the OAP upstroke into two components: the depth-weighted and the lateral-scattering, which corresponds to the components of the propagating electrical wave that are transmural (from endocardium to epicardium) and parallel to the epicardium. In this study the obtained data may have great value in evaluation of electrical signal formation and the formation of excitation in the heart under normal and pathological conditions.