Natural organic matter (NOM) components measured with ultrahigh-resolution mass spectrometry (UHRMS) are often assessed by molecular formula-based indices, particularly related to their aromaticity, which are further used as proxies to explain biogeochemical reactivity. An aromaticity index (AI) is calculated mostly with respect to carboxylic groups abundant in NOM. Here, we propose a new constrained AI based on the measured distribution of carboxylic groups among individual NOM components obtained by deuteromethylation and UHRMS. Applied to samples from diverse sources (coal, marine, peat, permafrost, blackwater river, and soil), the method revealed that the most probable number of carboxylic groups was two, which enabled to set a reference point = 2 for carboxyl-accounted AI calculation. The examination of the proposed AI showed the smallest deviation to the experimentally determined index for all NOM samples under study as well as for individual natural compounds obtained from the Coconut database. In particular, AI performed better than AI for all compound classes in which aromatic moieties are expected: aromatics, condensed aromatics, and unsaturated compounds. Therefore, AI referenced with two carboxyl groups is preferred over conventional AI and AI for biogeochemical studies where the aromaticity of compounds is important to understand the transformations and fate of NOM compounds.