) We present the development of a new heterogeneous immunoassay to determine the concentration ofhuman pancreatic lipase (HPL) using reflectometric interference spectroscopy (RIfS) and total internalreflection fluorescence (TIRF). HPL is a digestion enzyme used as a biomarker to detect an acute inflam-mation of the pancreas. The assay is carried out using the binding inhibition assay format. RIfS is used forantibody characterization and assay development as with this method binding signals can be observedtime-resolved. A good surface stability and reproducibility of the HPL biosensor can be realized. Usinga suitable regeneration agent makes it possible to perform up to 50 measurements on each RIfS trans-ducer. To achieve the required limit of detection, the immunoassay is transferred to the TIRF principleand is therefore the first HPL immunoassay based on fluorescence labeling of the antibody. A series ofconcentration measurements and their calibration show a sufficient working range of 0.068–3.849 mg/LHPL using TIRF. On each TIRF transducer, 20–30 measurements can be performed.