Substandard and falsified medicines have severe public health and socioeconomic effects, especially in low- and middle-income countries. The WHO has emphasized the need for reliable estimates of the prevalence of such medicines to efficiently respond to this problem. In the present study, we used 601 medicine samples collected in Cameroon, the DR Congo, and Malawi to assess the rates of substandard and falsified medicines based on different criteria. Based on the specifications of the U.S. Pharmacopoeia for the amount of the active pharmaceutical ingredients, the rate of out-of-specification medicines was 9.3%. By contrast, this rate ranged from 3.3% up to 35.0% if the tolerance limits of other pharmacopoeias or recently published medicine quality studies were used. This shows an urgent need for harmonization. Principal methods to assess the rate of falsified medicines are packaging analysis, chemical analysis, and authenticity inquiries. In the present study, we carried out an authenticity inquiry for the aforementioned medicine samples, contacting 126 manufacturers and 42 distributors. Response rates were higher for samples stated to be manufactured in Asia (52.4%) or Europe (53.8%) than for samples manufactured in Africa (27.4%; P < 0.001). One sample had been identified as falsified by packaging analysis by the local researchers and two additional ones by chemical analysis. Notably, seven additional falsified samples were identified by the authenticity inquiries. The total rate of falsified medicines resulted as 1.7%. Considerations are discussed for assessing the rates of "substandard" and "falsified" medicines in future medicine quality studies.