Abstract: The incidence and prevalence of onychomycosis are rising worldwide. Common diagnostic techniques often lack sensitivity or specificity. Differentiation between non-infectious nail disorders is frequently not possible. The aim of this study was to establish a better diagnostic routine procedure based on modern mass spectrometric peptide analysis techniques. One hundred and fifty-five nail samples from 145 patients with clinically suspected onychomycosis (n = 96, 62%) and without onychomycosis [e.g. nail psoriasis or nail dystrophy resulting from eczema (n = 59, 38%)] were investigated using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) peptide mass fingerprinting in comparison with standard techniques. We demonstrated that MALDI-TOF MS represents a precise, robust and fast tool in diagnostic investigation of nail disorders, which is superior to common standard methods.