A simple, automated and sensitive continues flow-injection analysis/merging zones technique (CFIA/MZ) method was developed for the determination of D-Penicillamine (thiol drug) in pure, pharmaceutical formulations and biological sample. This method involved the reaction of 1,2-Naphthoquinone-4-Sulphonic acid Sodium Salt (NQS) with D-Penicillamine to produce a brawn-colored complex which has maximum absorbance at 463nm. The method was cheap, economic, precise and accurate where the limit of detection was 6.3 µg.mL-1 and the RSD% was 0.34 and Recovery was 99.31%. Various chemical and physical conditions that affected the reaction have been studied. A wide calibration curve was rectilinear within the concentration range (15-1000) μg.mL-1 with a sample through put of 103 sample.hour-1. The proposed procedure was applied successfully for the estimation of PEN in pharmaceutics and biological sample and the results obtained were favorably compared with those given by a reference method of United States Pharmacopoeia (USP), and there was no significant difference between the obtained results, regarding accuracy and precision at the 95% confidence level.