Communities living in African animal trypanosomiasis (AAT) endemic areas of Zambia use several control strategies to protect their livestock from the devastating effects of trypanosomiasis. Several studies have reported the effectiveness of trypanosomiasis control strategies based on retrospective data. In this study, we assessed incidence rates of AAT in cattle ( = 227) using a prospective cohort study comprising 4 treatment groups, i.e., Diminazene aceturate, Isometamidium chloride, Cyfluthrin pour-on and Cypermethrin treated targets. The study was conducted in Mambwe district in Eastern Zambia between February 2019 and March 2020. The endemic prevalence of AAT for each group was determined using ITS-PCR prior to application of treatments. High endemic trypanosome pre-treatment rates were found in all Groups (Diminazene aceturate (61%), Isometamidium chloride (48%), Cyfluthrin pour-on (87%) and Cypermethrin targets (72%)). The overall apparent prevalence for the Mambwe district was 67% (152/227) and true prevalence at 95%CI was 63–71%. Once treatments were implemented, 12 monthly follow-ups were conducted. The average monthly incidence rates without standardization recorded: Diminazene aceturate (67%) Isometamidium chloride (35%), Cyfluthrin pour-on (55%) and Cypermethrin targets (61%). Incidence rates were standardized considering the endemic level of disease for each Group and the average standardized monthly incidence rate in the Diminazene aceturate Group was 7%; the Isometamidium chloride Group −13%; the Cyfluthrin Group −26%; and the Cypermethrin target Group, −17%. All Groups showed a decrease in incidence of AAT over the period of the study with the Cyfluthrin group showing to be the most effective in reducing AAT incidence in cattle.