Cell proliferation assays are an extensively employed tool to evaluate the efficacy of tested compounds on a biological ex vivo model of interest. In anticancer drug development, they are used to evaluate the anti-proliferative activity of tested compounds on established tumor cell lines, primary tumor cells and 3D tumor organoids (Adan et al., 2016; Finlay and Baguley, 1984; Horvath et al., 2016; Moffat et al., 2014; Boyd:1992ht Rello-Varona et al., 2015; Selby et al., 2017). In a typical assay, cells are plated on a culture vessel and, after a sufficient amount of time necessary to recover growing phase, tested compounds are added to culture medium. At arbitrary chosen time-points, the number of cells is estimated by cell count or by an indirect method, such as measuring DNA synthesis, lactate-pyruvate conversion or ATP concentration (Barone et al., 2017; Fiorentino et al., 2016; Kato et al., 2016; Marchesi et al., 2017; Nieddu et al., 2016; Pau et al., 2009). A parameter, representative of drug efficacy, is subsequently calculated for data representation. In this manuscript, we first describe the two most employed parameters to analyze raw data and represent in vitro drug efficacy: the relative cell number (R), used to calculate the half maximal inhibitory concentration (IC50), and the percentage of growth (PG), with an highlight on their limitations (Boyd and Paull, 1995; Yoshida et al., 1975). We emphasize that, using these parameters to compare drug efficacy between distinct cell populations (such as cell lines), cells that grow “faster” in culture will be inferred more sensitive than “slower” ones, and therefore these parameters lead to a misinterpretation of the results because of their dependency to the unique growth properties of each cell population. Despite PG partially overcomes this limitation, we provide in the first section of the results a detailed description of the dependency of R, and consequently the IC50, to cell proliferation rate because of its frequent usage in current anticancer drug discovery research (as few recent examples (Ben Jannet et al., 2017; Cabrera et al., 2017; Esposito et al., 2017; Hamed et al., 2017; Indovina et al., 2017; Koul et al., 2017; Mathema et al., 2017; Menderes et al., 2017; Mukunthan et al., 2017; Muñoz et al., 2017; Potenza et al., 2017; Ravi et al., 2017; Rimoldi et al., 2017; Said et al., 2017; Venkataramana Reddy et al., 2017; Wehbe et al., 2017; Zamora et al., 2017; Zhu et al., 2017)). Subsequently, in the second section of the results, we show that PG is also dependent on the growth properties of the cells, because of their exponential and not linear proliferation in culture.