Soil organic matter (SOM) plays a key role in the global carbon and nitrogen cycles. Soil biogeochemistry is commonly studied by extracting the base-soluble fractions of SOM: the acid-insoluble humic acid (HA) and acid-soluble fulvic acid (FA). Electrospray ionization-Fourier transform-ion cyclotron resonance-mass spectrometry (ESI-FT-ICR-MS) is commonly utilized for molecularly characterizing these fractions. Different sample preparation techniques exist for the analysis of HA and FA though questions remain regarding data comparability following different preparations. Comparisons of different sample preparation techniques revealed that the negative-mode ESI-FT-ICR-MS analytical window can be skewed to detect different groups of molecules, with primary differences in oxygenation, aromaticity, and molecular weight. It was also observed that HA and FA from soils versus an aquatic matrix behaved very differently and thus, we concluded that sample preparation techniques determined to be "most optimal" in our study are in no way universal, and we recommend that future studies of HA and FA involve such comparative studies for determining the most suitable sample preparation technique for their particular type of HA or FA matrices. This will enhance data comparability among different studies and environmental systems and ultimately, allow us to better understand the complex composition of environmental matrices.