A novel, extremely sensitive, first derivative spectrophotometric method has been developed for the simultaneous determination of nickel and cobalt with 5-(2’-carboxyphenyl) azoxine (R). R forms complex with the elements with overlapping spectra in aqueous medium at pH 5.2-6.1. The sensitization is achieved by use of non-ionic surfactant, Triton X-100. Color of the cobalt complex fades within 45 minutes, but the presence of nickel increases its stability appreciably making it difficult to determine nickel in presence of cobalt. The use of first derivative achieves selectivity to enable simultaneous determination of nickel and cobalt in presence of each other in the range 4.72 ng ml-1 to 235 ng ml-1 for nickel and 23.56 ng ml-1 to 235.6 ng ml-1 for cobalt by enhancing sensitivity of the already sensitized reaction with Triton X-100. The analytical characteristics of the analytical methods for the determination of nickel and cobalt compare favorably to that of ICP-OES. The method has been successfully applied to mixtures of nickel and cobalt in the varying ratios in the range studied, their alloys (in % range), and in house soil samples (in ppm range) with good precision and accuracy.