In recent years, the neglected diseases drug discovery community has elected phenotypic screening as the key approach for the identification of novel hit compounds. However, when this approach is applied, important questions related to the mode of action for these compounds remain unanswered. One of such questions is related to the rate of action, a useful piece of information when facing the challenge of prioritising the most promising hit compounds. In the present work, compounds of the “Leishmania donovani box” were evaluated using a rate of action assay adapted from a replicative intracellular high content assay recently developed. The potency of each compound was determined every 24 hours up to 96 hours, and standard drugs amphotericin B and miltefosine were used as references to group these compounds according to their rate of action. Independently of this biological assessment, compounds were also clustered according to their minimal chemical scaffold. Comparison of the results showed a complete correlation between the chemical scaffold and the biological group for the vast majority of compounds, demonstrating how the assay was able to bring information on the rate of action for each chemical series, a property directly linked to the mode of action. Overall, the assay here described permitted us to evaluate the rate of action of the “Leishmania donovani box” using two of the currently available drugs as references and, also, to propose a number of fast-acting chemical scaffolds present in the box as starting points for future drug discovery projects to the wider scientific community. The results here presented validate the use of this assay for the determination of the rate of action early in the discovery process, to assist in the prioritisation of hit compounds., Author summary Visceral leishmaniasis is a parasitic disease, caused by Leishmania donovani and Leishmania infantum species, found in Asia, South America, East Africa and Southern Europe. Currently available treatments present either serious side effects or high prices and, therefore, the identification of a new generation of antileishmanial drugs is clearly an unmet medical need. In order to find new and safer drugs, pharmaceutical companies and research institutes have engaged in screening of large compound collections on cell-based systems. These assays are more likely to predict the in vivo potency of the hits identified compared to assays based on the inhibition of a specific target, but do not provide any information regarding one of the most important parameters, together with PK/PD profile, used to predict the effect of a drug as it is the mode of action. In addition, most of the available assays rely on a single endpoint determination of compound potency and, therefore, do not evaluate rate of action, a property linked to the mode of action. Although there is only limited knowledge on the PK/PD profile for current antileishmanial drugs amphotericin B and miltefosine, the observation that in vitro fast-acting amphotericin B requires shorter treatment regimen than in vitro slow-acting drug miltefosine seems to suggest that this property could be very useful for progressing the most promising hit compounds to more advanced stages of the drug discovery process. In fact, progression of fast-acting compounds over slow-acting ones is the current strategy at the Kinetoplastid DPU. For these reasons, we present here the application of a rate of action assay to a promising compound set displaying potencies against the intracellular Leishmania donovani parasite (”Leishmania donovani box”) in the micromolar to sub-micromolar range as well as its validation for use in the hit prioritisation within drug discovery.