Phytohormones are structurally diverse small molecules that regulate plant developmental processes. Correct activity depends on strict cell and tissue specific accumulation, and analysis of phytohormone localization is crucial for understanding their mode of action and possible utilization in crop development. Today, this is achieved by either dissection experiments that are cumbersome and possibly sub-optimal in terms of resolution, or by indirect detection methods that do not always fully reflect phytohormone distribution. They may furthermore require genetic transformation and hence only work for model plants and a limited number of crops. Mass spectrometry imaging (MSI) visualizes the distribution of small molecules across a tissue sample, at a typical resolution of 5-50 µm. Hence, MSI can potentially alleviate some issues with spatial phytohormone analysis and provide new insights into their distribution and biology. However, like other organisms, plants accumulate hormones in extremely low amounts, challenging their detection in MSI. The newly developed MALDI-2 (Matrix Assisted Laser Desorption Ionization) post-ionization technology can enhance molecular ionization efficiency, potentially increasing sensitivity for detection of lowly abundant metabolites. The success of MALDI-2 enabled enhanced sensitivity depends on molecular characteristics of the analytes as well as the ionization matrix. We tested a range of phytohormones and matrices in co-crystallization experiments to find combinations that achieved enhanced ionization and detection sensitivity for several phytohormones. We furthermore optimized parameters for imaging phytohormone distributions in plant tissues and show the successful direct imaging of auxin distribution in sections of pea plants undergone different treatments to induce changes in auxin localization.