Water samples were taken in Hidden Lake at five different time points around two rotenone applications: (i) five weeks prior to the first rotenone application, on July 12 2018; (ii) approximately three weeks after the first application of rotenone, on 7 September 2018; (iii) approximately 10 months after the first rotenone application, on 10 July 2019; and (iv) one year after the final rotenone treatment, on 19 August 2020. For each time point, four pelagic and four littoral water samples were taken from Hidden Lake, as well as 8 to 13 water samples from Hidden Creek and Coral Creek for a total of 16 to 21 samples per time point. Quantitative PCR (qPCR) method was used to produce brook trout (Salvelinus fontinalis) cytochrome b copy number for each sample. The objective of this study was use eDNA to assess the efficacy of invasive brook trout removal using rotenone.