A Decade of Decline: The Decreasing Use of Positive Controls Threatens the Reliability of In vitro Cancer Research

Positive controls are indispensable for validating in vitro cancer bioassays and ensuring reproducibility in cancer drug discovery research. However, evidence suggests their inclusion in in vitro bioassays during drug discovery is declining, undermining assay reliability, reproducibility, and scientific integrity. This mini-review analysed 150 peer-reviewed studies published in 2015, 2020, and 2025 (50 from each year) to evaluate trends in positive control usage within cytotoxicity assays targeting cancer cell lines. Results indicate that only 52% (2015) and 54% (2020) of studies included appropriate positive controls; however, this dropped to a worrying 32% in 2025. The omission was most pronounced in natural product research and studies using high-throughput assays, such as MTT. In addition, reporting of dose-response parameters (e.g., IC₅₀) decreased from 26% to only 16% across the decade. These findings highlight a systemic erosion of methodological rigor, likely driven by publication pressure, limited resources, and a lack of standardised protocols. We recommend the mandatory inclusion of validated positive controls, transparent reporting of preparation and storage conditions, and journal-level enforcement of experimental standards. Reinforcing these practices will improve reproducibility, comparability, and ultimately the translatability of preclinical cancer research outcomes.