A room temperature operated pulsed external-cavity (EC) quantum cascade laser (QCL) was used for mid-infrared (mid-IR) transmission measurements of glucose and lactate in aqueous solution. The high spectral power density of the EC-QCL (ranging from 1–350 mW) over a wide tuning range (1030–1230 cm−1) allowed transmission measurements through optical paths of 130 μm and more. This is a significant improvement in terms of robustness of the measurement setup, especially when samples containing cells or other particles, as is the case for biofluids, are to be analyzed. The broad tuning range furthermore permitted multi-analyte detection based on multivariate calibrations. Promising results on the simultaneous determination of glucose (c = 0–800 mg dL−1) and sodium-lactate (c = 0–224 mg dL−1) in aqueous solutions in the presence of the interferents maltose and xylose are reported. A partial least squares (PLS) calibration model was calculated which was able to predict the glucose concentration with a root mean square error of prediction (RMSEP) of 9.4 mg dL−1, as proved by external validation. Due to their small size and room temperature operation, EC-QCLs offer an attractive alternative regarding the way mid-IR measurements are carried out. This may be of special importance for new reagent-free bedside monitoring systems.