Naphthenic acid (NA) fractions from heavy crude oils are typically analyzed in mass spectrometry via electrospray ionization (ESI). Here, we show that NA analysis is also possible by MALDI using a highly basic matrix (proton sponge). When compared with ESI, that allowed detection of six compound families, MALDI resulted in the observation of twenty-two compound families. Besides, MALDI analysis affords compositional-dependent information. Unlike ESI, where all NAs are detected as deprotonated molecules, in MALDI we observe both deprotonated molecules and radical anions. This indicates that ion formation in NA by MALDI can occur by either acid-base and electron-capture mechanisms, or both. Interestingly, the preferred ionization channel seems to be compound class dependent; for instance, the acid base mechanism (which allows detection of deprotonated NA species through a Bronsted-Lowry reaction) applies exclusively to NAs with high H/C ratios along the whole O/C range. On the other hand, for less saturated compounds both mechanisms apply, while the electron capture channel occurs exclusively in species with low H/C and high O/C ratios or high H/C and low O/C ratios (envelope of the van Krevelen plots). In summary, we demonstrate that MALDI(-) is an alternative method for the analysis of complex petrochemical samples offering additional advantages, when compared with ESI, such as increasing compositional space accessibility for polar samples, high throughput and exceptional tolerance to the presence of impurities and salts in the samples. Furthermore, exploration on semi-quantitative approaches in MALDI can provide additional advantages such as those offered by LC-ESI MS, currently used to quantify naphthenic acids.