In the field of medical microbiology, matrix-assisted laser desorption ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) secures a rapid identification of clinical isolates. This PhD thesis aimed at implementing MALDI-TOF MS to the characterization of soil bacteria called rhizobia, which form nitrogen-fixing symbioses with plants of the Leguminosae family. Dedicated mass spectral databases and protocols for the rapid characterisation of symbiotic rhizobia were developed. Together these tools allow for a cost-effective high-throughput screening of rhizobia directly from plant tissues instead of cultured bacterial isolates. Taking advantage of the rapidly expending genomic data, we also showed that empirical mass spectra can be replaced by sets of sequence-deduced ribosomal proteins and that plant leghemoglobins are reliable markers for identifying host plants. MALDI-TOF MS has now become a tool of choice to probe the diversity of rhizobia and symbiotic associations that exist in nature, or develop new bio-inoculants for agronomy.