This work aims to demonstrate the potential of pulsed laser ablation synthesis (PLA) of tellurium nanoparticles (Te NPs) for use in matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS) applications. An experimental laboratory setup for PLA synthesis of fresh Te NPs was designed to prevent unwanted aggregation of uncoated Te NPs and avoid the need to use additional modifiers. Performing pulsed laser ablation synthesis in liquid (PLAL) using acetone was found to be the optimal way of preparing Te NPs. Another possibility is to use commercially available laser ablation devices for laser ablation - inductively coupled plasma mass spectrometry (LA-ICP-MS) to perform PLA in a helium atmosphere, but this approach is less efficient and results in the formation of unwanted larger particles. The prepared Te NPs were studied using the transmission electron microscopy (TEM) and dynamic light scattering (DLS) methods. TEM images showed the formation of Te NP nanochains composed of many crystallized Te NPs with sizes ranging from 8 to 15 nm. The various size distributions of the synthesized Te NPs identified using the DLS method correspond to the size distributions of aggregations rather than individual Te NPs. The synthesized Te NPs were used for a pilot study of their possible use with the MALDI-MS technique. An important effect was observed when Te NPs were used to perform a MALDI-MS analysis of the alpha-cyclodextrin ( alpha-CD) and cucurbit[7]uril (CB7) macrocycles, which consisted in a decline in the formation of matrix adducts. Furthermore, several changes in MALDI-MS mass spectra of intact cells and a positive effect of Te NPs on the crystallization of the MALDI-MS matrix were observed.