Methanol is an organic compound commonly used as a solvent in the synthesis of fuel for internal combustion engines. However, the adulteration of fuels (biofuels and gasoline) with methanol is commonly practiced throughout world, and regulations to control the content of methanol in these fuels have been imposed. Although methanol in fuels can be analyzed by numerous analytical techniques, the traditional methods have some disadvantages, such as interferences, incomplete reactions, and lengthy analysis. Thus, the present work proposes a rapid and versatile methanol determination method using a simple derivatization of the sample with nicotinoyl chloride and subsequent fast analysis by ambient mass spectrometry using venturi easy ambient sonicspray ionization (V-EASI-MS), without preseparation, taking into account all parameters of linearity, limits of detection, quantification, accuracy, and precision based on ISO 5725. We were able to develop a powerful method to quantify methanol in fuels which can be used as an alternative method to detect adulteration by government agencies.