Bacterial lysates have for long been used to boost the immunological response to Respiratory Tract Infections (RTI) both in children and adults. They are prepared by growing bacteria usually associated with RTI, followed by chemical or mechanical disruption to prepare single bacterial lysates that are combined in the final product. Despite the wide range of applications, one drawback for their universal use is the difficulty to assure consistency in their composition given their particular form of preparation; thus there is a need for alternative analytical methods that ensures batch composition consistency. Here, we demonstrate that MALDI-TOF MS provides reliable and reproducible mass spectral fingerprints for bacterial lysates of S. pneumoniae, H. influenzae, K. pneumoniae, Staphylococcus spp. We also found that mechanical disruption provides markedly betterdefined fingerprints. Analysis of the formulated Polyvalent Bacterial Mechanical Lysate (PBML) also showed a characteristic spectrum. Overall, we found that mechanical lysis coupled to MS analysis allowed for accurate and highly sensible detection of key proteins in each bacterial lysate, a method that can be used to standardize batch-to-batch product composition. Applying this methodology to the production pipeline shall result in better products expanding the acceptance of these cost-effective tools to prevent respiratory tract pathologies.