In the present scenario of drug discovery, several screening filters ensure a rigorous nomination of clinical candidates. One of these screens is the determination of IC50, the concentration of drug at half-maximal inhibitory concentration, also known as a potency assay. However, various nuances pertaining to the design, execution, and interpretation of in vitro potency results suggest a sizeable opportunity for the generation of erroneous data. The focus areas of this article include: (1) examining the requirement for the addition of serum albumin in in vitro potency assays, (2) problems encountered with cell lysates, and (3) drug candidate stability concerns during in vitro potency assays/high-throughput screening. Based on this assessment, the interpretation of the data generated using cell-based systems (i.e., lysates with or without the addition of fetal bovine serum) should be carried out with caution for in vitro potency testing, and the inclusion of a correction factor for non-specific protein binding should be considered. The addition of serum albumin to a cell-free system should be restricted to drugs having high protein binding (≥ 90%). Additionally, stability assessment of analytes should be considered to avoid dubious in vitro potency outcomes due to degraded material or active metabolite(s).