The increasing global production and application of nanomaterials for biological and industrial purposes have led to their release into the environment, raising ecological and public health concerns. Nanoparticles (NPs) present in the environment may be absorbed by plants and other biological systems. Analytical approaches that provide information on the spatial distribution of NPs in plant materials are essential for understanding the mechanisms of nanoparticle mobility in biological systems. Recent developments in Single Particle-Inductively Coupled Plasma Mass Spectrometry (SP-ICP-MS) have enabled the assessment of NP biotransformation in plant tissues. However, since SP-ICP-MS requires NPs in solution, enzymatic digestion is often used, which can compromise spatial information and affect the integrity of NPs. Laser Ablation−Single Particle−Inductively Coupled Plasma−Mass Spectrometry (LA-SP-ICP-MS) has emerged as a cutting-edge method for direct solid sampling, providing enhanced insights into the size, concentration, and spatial distribution of NPs within solid matrices. In this study, a gelatin-based microdroplet calibration method was developed for the quantitative mapping of AuNPs in plant tissues using LA-SP-ICP-TOF-MS. Porcine gelatin standards spiked with commercial AuNPs (30 nm, 50 nm, and 100 nm) were prepared in 10% gelatin, with Indium (In) as the internal standard. Microdroplets ranging from 0.015 to 0.100 mg were manually deposited onto microscope slides and analyzed using a laser ablation system optimized for microdroplet ablation and minimal NP degradation. Laser energy densities from 0.1 to 1.8 J/cm² were tested, and firing 50 pulses at a fluence of 0.25 J/cm² produced reliable results. To assess transport efficiency (TE), gelatin standards were digested, and NP counts from LA-SP-ICP-TOF-MS were compared with bulk analysis by ICP-MS; TE ranged from 95-105%. As a proof of concept, the method was applied to map AuNPs in bean cotyledon cross-sections as well as essential elements (Cu, Fe, Mn, and Zn).