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N-containing heterocycles (NCHs) are largely used as precursors for pharmaceuticals and can enter the environment. Some NCHs have been shown to be toxic, persistent, and very mobile in the environment. Thus, they have received increasing attention in the past years. However, the analysis of these polar compounds in environmental samples is still a challenge for liquid chromatography. This paper investigates the use of mixed-mode liquid chromatography (MMLC), which has reversed-phase and ion exchange characteristics for measurements of NCHs in water. NCHs with low pKa (i.e., < 2.5) display mainly reversed-phase interactions (neutral species) with the stationary phase and those with higher pKa (i.e., > 5) interact by a mixture of reversed-phase/ion exchange/HILIC mechanism. It was also shown that the presented method performs well in the quantification of the majority of the selected NCHs in surface water with MDLs between 3 and 6 μg/L, a low matrix effect and recoveries in the range of 77–96% except for pyridazine exhibiting 32% were achieved. The method was successfully employed to follow the degradation of NCHs in ozonation.