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The search for more suitable alternatives in analytical processes is a strategy to reduce environmental damages. The present study aimed to quantify the soil microbial biomass carbon (SMB-C), establishing a comparison between the methods involving quantification of SMB-C by titration of the samples with ferrous sulfate ammonia and molecular absorption spectrophotometry methods. This comparison was performed by two soil sample tests: (i) areas of grain crops with conventional management versus notill farming; and (ii) areas with distinct phytophysiognomies in the region southern Mato Grosso do Sul state (Cerrado and Semideciduous Forest). It was found that molecular absorption spectrophotometry was an efficient tool for the determination of soil microbial biomass carbon, allowing replacement of the titrimetric method. There were significant differences in the levels of SMB-C determined spectrophotometrically in relation to those determined by titration. However, for the levels of SMB-C determined by spectrophotometry to be compared with those determined by titration, the values must be corrected by the linear regression equation Y spectrophotometry = - 151.38 + 0.92532 * X titration . Key words : Potassium dichromate, soil quality, titration.