The aim of this study was to demonstrate that mid-infrared spectroscopy is able to quantify glucose in a serum matrix with sample volumes well below 1 muL. For this, we applied mid-infrared attenuated total reflectance (ATR) or transmission-based spectroscopic methods to glucose quantification in microsamples of dry-film sera, either undiluted or diluted 10 times in distilled water. The sample series spanned physiological glucose concentrations between 50 and 600 mg/dL and volumes of 80, 8, and 1 nL. Calibration was carried out using multivariate partial least-squares (PLS) modeling with spectral data between 1180 and 940 cm(-1). Best performance was achieved in the ATR experiments. For raw ATR spectra, the optimum standard error of prediction (SEP) of 13.3 mg/dL was obtained for the 8 nL sample series with subsequent 10-fold dilution. With respect to the coefficient of variation of the glucose assay, CV(pred), we obtained a value of 3% for the 80 nL volume samples with spectral preprocessing using matrix protein absorption bands as an internal standard, 4% for the 8 nL samples, and 6% for the 1 nL samples with raw data. Spectral standardization resulted in significant improvement, especially for the 80 nL volume sample series. By contrast, the accuracy of the glucose assay for the 1 nL sample volume series could not be improved either by internal standardization or by considering the dry film areas for normalization, which we attribute to varying topographies of the dry films.