During the reproductive stage, tomatoes require nighttime temperatures below 21°C and daytime temperatures below 32°C for healthy flower development and fruit set. Prolonged exposure to temperatures above the optimal range can cause abnormalities in pollen development, leading to reduced fertility or sterility. This project aims to identify alterations in proteome expression and elucidate heat tolerance mechanisms affecting pollen development under elevated temperature conditions. Two tomato varieties—the heat-tolerant ‘Black Vernissage’ and the heat-sensitive ‘Micro-Tom’—were exposed to three temperature treatments: 26 °C (control), 32 °C, and 37 °C (heat-treated). Flower buds containing meiotic pollen mother cells prior to the tetrad stage and free microspores were harvested, and single cell type samples were collected using laser microdissection. Tandem mass tag (TMT) proteomics was employed to identify differential proteomic responses in pollen mother cells and free microspores between stress-treated and non-treated plants.