Biohydrogen production utilizing negative valued waste through dark-fermentation process is one of the emerging areas. Reported conditions for H-2 production are significantly variable and comparative analysis of data is major problem for unified understanding. A simple, rapid and generalized two phase methodology/protocol was developed to evaluate the biohydrogen production potential (BHP) of negative valued wastewater as substrate/feed-stock for renewable biohydrogen production using mixed consortia. Critical factors that can influence the overall process viz., redox condition, organic load and biocatalyst were considered in the designing the methodology. Feasibility of protocol was initially evaluated with synthetic wastewater and further validated with real field composite food and slaughter house wastewaters. The selected operational factors showed marked influence on both H-2 production and wastewater treatment. The reported methodology/protocol not only provides the ability of selected wastewater to generate H-2 but also facilitates process understanding based on selected factors and finally acquiesce optimum conditions. Copyright (C) 2011, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.