2-hydroxy-1-naphthaldehyde-p-hydroxy benzoic hydrazone (HNHBH) is used as a new analytical reagent for the direct and derivative spectrophotometric determination of vanadium (V). It reacts with vanadium in acidic medium (pH 4.0, sodium acetate and acetic acid buffer) to form deep yellow colored (λ max, 430 nm) 2:3 (V- HNHBH) complexes. The colour reaction is instantaneous and the absorbance remains constant for about 72h. The molar absorptivity and Sandall’s sensitivity were found to be 2.30 x 104 L mol-1cm-1 and 0.0025 μg cm-2 respectively. The system obeys Beer’s law in the range of 0.101-1.121 μg mL-1 of V (V). Tolerance limits of various foreign ions were also studied. The complex was fairly stable (stability constant 4.73 x 1019) and Second and third order derivative spectrophotometric methods were also developed for the determination of vanadium (V) which showed greater sensitivity and selectivity. The proposed method was applied for the determination of vanadium (V) in Different Environmental, Leafy vegetable, and Biological Samples. The method has high sensitivity, selectivity, and precision and accuracy.