Nicotine is an alkaloid that can be naturally found in several edible plants of certain families such as the nightshade family. While nicotine has been also reported in other edible plants such as mushrooms without any clear scientific consensus about their origin. Hence, this study aimed to develop a method for the determination of nicotine in mushrooms. Nicotine is a very analytically challenging compound with two pkas and three forms (double ionized, single ionized, and non-ionized). Therefore, this study aimed to utilize nicotine’s unique characteristic for extraction optimization and chromatographic separation. The optimum conditions were chosen based on matrix effect, spike recovery and chromatographic separation. The final method involved extraction using methanol under alkaline conditions (5% ammonium hydroxide). Alkaline condition in injection solvent was optimum for separation in reversed liquid chromatography. Additionally, the spiking recoveries were comparable with the acidic conditions, with the matrix effect being in favor of the alkaline conditions. The developed protocol was validated in accordance with the method validation criteria stated in the document SANTE/11312/2021 (V2) in terms of linearity, matrix effect, trueness, precision, and limit of quantification. A small-scale survey of six samples were done on mushrooms from the Egyptian market, all samples were found to be under permissible limits.