Work-related exposure to bio-allergens, such as fungal α-amylase, is a well-known cause of occupational rhinitis and asthma. Therefore, it is important to have suitable analytical methods available so that exposure reduction measures can be taken when levels are too high. In this study, concentrations of airborne fungal α-amylase down to 5 ng/filter were determined by means of a new analytical method that is based on enzymatic digestion, using trypsin, followed by liquid chromatography (LC) and high-resolution accurate-mass (HR-AM) mass spectrometry (MS) with targeted selection of enzyme fragment ions of interest (tSIM). Quantification was performed using an external calibration curve based on a fungal α-amylase from Aspergillus niger (Fungamyl Ultra^®). The manufacture of this enzyme and the acquisition of the analytical samples by air sampling and subsequent collection on PTFE filters took place at the same time at the same enzyme production facility. By means of the new HR-AM tSIM LC-MS method, the magnitude of occupational exposure can be elucidated.