New methods for spectrophotometric alkalinity measurement, dissolved hydrogen monitoring and for obtaining a fingerprint of the microbial community were evaluated as tools for process monitoring in anaerobic digestion. The anaerobic digestion process was operated at organic loading rates of 1.5, 3.0 and 4.5 g volatile solids l(-1)d(-1) and subjected to pulse loads of carbohydrate, lipid, protein and a mixed sludge substrate. The spectrophotometric alkalinity monitoring method showed good agreement with traditional titrimetric alkalinity monitoring and has the advantage of being easy to modify to on-line monitoring applications. The on-line monitoring of dissolved hydrogen gave valuable information about approaching process overload and can be a good complement to the conventional monitoring of volatile fatty acids. Changing process conditions were also reflected in the microbial fingerprint that could be achieved by partitioning in two-phase systems. The investigated methods showed potential for application in increasing our understanding of the anaerobic digestion process as well as for being applicable for monitoring in the complex environment of full-scale anaerobic digestion processes.