Sublimation is one of the preferred methods of choice for matrix deposition in high spatial resolution MALDI mass spectrometry imaging (MALDI-MSI) experiments. However, reproducibility and time are the major concerns for this setup. Here we present a lab-made glass sublimator with significant improvements in fine control of the vacuum with real-time monitoring and a rapid sublimation process of only 22 min. This method yielded reproducible homogeneous matrix crystals of <1 μm on the sample surface. MALDI-MSI was performed in tissue sections of barley inflorescence meristems at 15 μm spatial resolution, thus demonstrating its efficiency. Overall, we believe these simple yet effective new modifications can be easily adapted to the standard glass sublimation devices to achieve highly reproducible matrix deposition for high spatial resolution MALDI-MSI.