Simple SummaryThe “WHO susceptibility bioassay” is a method from the World Health Organization used to monitor the resistance to insecticides in mosquito populations. This method was first developed in the 1960s and has undergone multiple changes since then. While these changes may appear minor, the numerous iterations of the test procedures leave some parameters open to interpretation, and changes to methodology may affect results. To address this, we reviewed the published test procedures for this method and the published literature which cited this method to see where the method could be optimized and exactly how people were reporting their use of this method. This revealed that the method is not being carried out consistently, and that the most up to date iterations of the test procedures are not always referenced. To address this, recommendations on the referencing and reporting of this method were developed. Alongside this literature review, we detail experimental work that explored whether altering parameters with room for interpretation in the test procedures could impact bioassay results. From the results, suggestions have been made to tighten certain parameters to avoid inaccurate measures of insecticide resistance. Closer adherence to the method and tightened parameters should lead to the generation of more robust data from the bioassay.Accurately monitoring insecticide resistance in target mosquito populations is important for combating malaria and other vector-borne diseases, and robust methods are key. The “WHO susceptibility bioassay” has been available from the World Health Organization for 60+ years: mosquitoes of known physiological status are exposed to a discriminating concentration of insecticide. Several changes to the test procedures have been made historically, which may seem minor but could impact bioassay results. The published test procedures and literature for this method were reviewed for methodological details. Areas where there was room for interpretation in the test procedures or where the test procedures were not being followed were assessed experimentally for their impact on bioassay results: covering or uncovering of the tube end during exposure; the number of mosquitoes per test unit; and mosquito age. Many publications do not cite the most recent test procedures; methodological details are reported which contradict the test procedures referenced, or methodological details are not fully reported. As a result, the precise methodology is unclear. Experimental testing showed that using fewer than the recommended 15–30 mosquitoes per test unit significantly reduced mortality, covering the exposure tube had no significant effect, and using mosquitoes older than 2–5 days old increased mortality, particularly in the resistant strain. Recommendations are made for improved reporting of experimental parameters