A technique for modification of the microtiter reader plates well with a polyaniline (PANI) film sensitive for ascorbic acid is presented. The principle of the analyte detection is based on monitoring the changes in optical absorption of the PANI film resulting from the reduction process initiated by ascorbic acid. The detection limit for ascorbic acid is 1 mg/L. Testing with real samples (soft drinks, fruit juices) gave good correlation of the method with iodimetric titration. High sensitivity, stability, and good reproducibility of the measurements make the proposed system an attractive alternative to traditional assays, used in medicine, ecology, and biotechnology.