“Cellulosomes” produced by certain strains of anaerobic bacteria are very large and complex supramolecular “machines” that are extremely efficient in catalysis of depolymerization of cellulose to soluble sugars. Our experimental results emphasize the extent to which quantitative assessment of cellulosome activity is affected, in ways by no means completely understood, by a wide variety of factors that can be roughly grouped into those factors altering the composition of the cellulosome complex itself, and those assay conditions affecting activity and survivability of cellulosome components. Cellulosome composition may vary with the conditions under which the organism produces the complex and with the procedure used to separate the “cellulosomal” fraction from other proteins. Enzyme activity and survivability in assay are strongly affected by highly interactive choices of assay conditions such as nature of substrate used, pH, temperature, redox potential, enzyme loading, assay duration, extent of conversion of substrate measured, and accumulation of inhibitory products such as cellobiose. Our laboratory is investigating the effects of these factors on assay results using two different categories of “cellulosomal” preparations, one category being “native” cellulosomal fractions produced and purified under a variety of conditions, the other category being genetically-engineered “designer minicellulosomes” containing selected, defined arrays of recombinant catalytic, substrate-binding, scaffoldin and linker domains. Findings from this large experimental array are presented and discussed both in terms of assay-development and implications for fundamental understanding of cellulosome action.