Drug discovery has historically relied on the massive screening of compound libraries with in vitro cell-based target assays. The challenges of performing larger screening campaigns while keeping time and costs limited has led the pharmaceutical industry to develop in vitro high-content screening (HCS) technologies. These techniques, generating thousands of data points per day, require standardized and robust data analysis procedures that can enable a rapid decision-making process. Quality control (QC), processing, and reporting are the typical steps applied to experimental HCS data. During QC sessions, all images’ control parameters are inspected against standard acceptance criteria. Processing includes data normalization, dose-response fitting, and evaluation of standard parameters, such as EC50 and minimal effective concentration (MEC). Analysis results are then shared via formatted reports. Deficient analysis pipelines lead to long, untraceable procedures. GladiaTOX is an open-source solution for HCS data processing and reporting that expands the tcpl package (toxcast pipeline, Filer et al. 2016). In addition to tcpl’s functionalities (multiple dose-response fitting and best fit selection), GladiaTOX (i) fetches raw image quantifications via a webservice layer, also allowing multiple (proprietary) systems to be integrated easily; (ii) computes MEC based also on historical data; (iii) exports results formatted for ToxPI GUI; (iv) aggregates single endpoint MECs into severity scores; and (v) implements a suite of functionalities for QC reporting to a structured PDF format. The effects of seven chemical constituents present in tobacco or tobacco smoke on normal human bronchial cells are reported as use case: three metals (arsenite, mercury, and selenite), two phenols (phenol and p-cresol), acrylamide, and naphthalene. Overall, metals show low MEC values (high toxicity) on most endpoints tested (including cytotoxicity assays, DNA damage, and oxidative stress). The severity scores rank metals as more toxic than phenols and assign low severity to acrylamide and naphthalene. GladiaTOX represents an all-in-one, open-source, flexible solution to store, process, and report HCS data in biomedical research.