The nonperoxide antibacterial activity of New Zealand ma̅nuka honey originates from dihydroxyacetone (DHA) within Leptospermum scoparium nectar. This study determined if DHA was present within the nectar of four Australian Leptospermum species: L. laevigatum, L. polygalifolium, L. trinervium, and L. whitei. A rapid and convenient new method was developed, which quantitated DHA/sugar ratios (ppm). The DHA and sugars were derivatized with o-(2,3,4,5,6-pentafluorobenzyl) hydroxylamine hydrochloride and analyzed via RP-HPLC with diode array detection at two wavelengths (200 and 243 nm). DHA was detected in all L. whitei and L. polygalifolium samples, where DHA/sugar ratios ranged from 10169 to 24199 ppm and from 9321 to 20174 ppm, respectively. DHA was undetected in any of the L. laevigatum and L. trinervium samples, and nectar activity was <100 ppm. The results of this study have implications for the Australian beekeeping industry, as the findings indicated that not all species of Leptospermum will produce active honey.