Trembling aspen (Populus tremuloides Michx) bark was extracted with water and the crude extract fractionated with tert-butyl-methyl ether (TBME), ethyl acetate (EtOAc) and n-butanol (BuOH) to obtain four different fractions. The antioxidant properties of the bark hot water extracts and its fractions were determined by three in vitro experiments: DPPH assay, phosphomolybdenum assay and canola oil thermoxidation assay by DSC analysis. Most of the results of the reported tests showed that the crude hot water extract and its fractions exhibited a strong antioxidant activity, higher than the synthetic antioxidant BHT. The results of this study confirm that antioxidant activity is a property that strongly depends on the oxidation conditions used in the particular oxidation test. Among the fractions separated from the aqueous extracts of bark, BuOH, TBME and EtOAc soluble fractions exhibited the best antioxidant efficiency, in phosphomolybdenum assay, DPPH assay and canola oil thermoxidation, respectively. Total phenol, flavonoid and flavanol contents were also evaluated and the results confirmed that the polyphenols contained in the hot water extract of this bark are mainly composed of non-flavonoid compounds.