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Matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) based microbial identification is rapid, cost-effective and proved to be as reliable as genome based methods. Either an intact microbial colony or the whole protein extract is utilized to obtain a MALDI spectra and the species identification is achieved by matching the spectra with the reference spectra library. In recent years, it has been demonstrated that MALDI can be extended for differentiation/species identification of immune cells, insects, ticks and fish. Since the current commercial reference libraries are limited to the microorganism, we utilized a simple formic acid/acetonitrile extraction procedure to establish a reference library for generally accepted species of the micro-algae Prototheca spp., the only known infectious algae. Following which we have successfully implemented MALDI in place of expensive PCR analysis for the routine identification and diagnosis of Prototheca in our laboratory. More recently, we utilized this simple sample preparation technique to compile a reference database for different body parts of the insect Tsetse fly (Glossina) - biological vector of African sleeping sickness causing trypanosomes. Our work and other recent publications highlight that inclusion of additional reference spectra enables rapid and universal identification of microorganisms, insects and other higher organisms of veterinary importance. The future research focus is to establish a reference library that could enable single extraction procedure and one MALDI measurement to provide information such as species, presence of pathogens and antimicrobial resistance.