Neutrophil granulocytes are critical mediators of innate immunity and tissue regeneration. Rare diseases of neutrophil granulocytes may affect their differentiation and/or functions. However, there are very few validated diagnostic tests assessing the functions of neutrophil granulocytes in these diseases. Here, we set out to probe omics analysis as a novel diagnostic platform for patients with defective differentiation and function of neutrophil granulocytes. We analyzed highly purified neutrophil granulocytes from 68 healthy individuals and 16 patients with rare monogenic diseases. Cells were isolated from fresh venous blood (purity >99%) and used to create a spectral library covering almost 8000 proteins using strong cation exchange fractionation. Patient neutrophil samples were then analyzed by data-independent acquisition proteomics, quantifying 4154 proteins in each sample. Neutrophils with mutations in the neutrophil elastase gene ELANE showed large proteome changes that suggest these mutations may affect maturation of neutrophil granulocytes and initiate misfolded protein response and cellular stress mechanisms. In contrast, only few proteins changed in patients with leukocyte adhesion deficiency (LAD) and chronic granulomatous disease (CGD). Strikingly, neutrophil transcriptome analysis showed no correlation with its proteome. In case of two patients with undetermined genetic causes, proteome analysis guided the targeted genetic diagnostics and uncovered the underlying genomic mutations. Data-independent acquisition proteomics may help to define novel pathomechanisms in neutrophil diseases and provide a clinically useful diagnostic dimension.