New synthesized reagent 2,6-Diacetylpyridine bis-4-phenyl-3-thiosemicarbazone (2,6-DAPBPTSC) is proposed as a sensitive and selective analytical reagent for the separation and extractive spectrophotometric determination of palladium(II) at pH: 4.0 to form a yellowish orange colored 1:1 chelate complex, which is very well extracted in to isoamyl alcohol. The maximum absorbance was measured at 410 nm. This method obeys Beer’s law in the concentration range 0.0 -12.6 mg mL-1 and the correlation coefficient of Pd(II)- 2,6-DAPBPTSC complex is 0.962, which indicates an adequate linearity between the two variables with good molar absorptivity and Sandell’s sensitivity, 1.156 ´ 104 L mol-1cm-1 and 0.0092 mg cm-2, respectively. The instability constant of complex calculated from Asmus’ method is 1.667 ´ 104 at room temperature. The precision and accuracy of the method is checked with calculation of relative standard deviation (n = 5), 0.371. The method was successfully applied for the determination of Pd(II) in spiked samples. The performance of present method was evaluated in terms of Student‘t’ test and Variance ratio ‘f’ test which indicates the significance of present method over reported method.