Background: Scenedesmus sp. AMDD (S-AMDD) has been the focus of several studies to assess its potential as a feedstock for biofuels and bioremediation, while the evaluation of its potential suitability as a novel animal feed ingredient has only ju st begun. In an initial study; S-AMDD demonstrated rapid growth rate and biomass productivity during exponential growth phase and the resulting biomass appeared to have good potential for animal nutrition based on its attractive proximate composition, favorable essential amino acid, fatty acid and elemental profiles and lack of contaminating heavy metals. However, the total carbohydrate and fibre fractions of whole-cell and lipid-extracted S-AMDD were relatively high which could limit their digestion, particularly when fed to monogastric animals. The difference in the capacity to digest and metabolically utilize diets rich in cellulosic material (e.g., fibre) is vast between various farmed animal species. As such, knowledge on the nutritional value of novel ingredients for ruminant animals can rarely be immediately extrapolated to monogastrics and vice versa. Simulated fermentation using rumen-derived digestive fluids or in vitro digestibility using purified monogastric-derived enzymes can provide valuable information. Although not fully conclusive, results from these types of rapid assays are generally in expensive, require smaller amounts of sample; utilize fewer (or zero) experimental an imals, avoid feed refusal issues associated with ingredient off-flavours or odours and can be effective tools for research and for routine industrial use. Objective: The present study is the second in a series of projects designed to evaluate the nutritional value of S-AMDD for animal feed applications. The main objective was to generate novel digestibility data of whole-cell and lipid-extracted S-AMDD for both ruminant an d monogastric animals including ruminal organic matter digestibility (OMD), apparent metabolizable energy (aME) content, methane (CH4) production, dilute pepsin digestibility (DPD) and two-phase gastric/pancreatic digestibility of protein (GPDProtein) and energy (GPDEnergy). Methods: Simulated ruminal OMD, aME contents and CH4 production of experimental test diets containing graded levels of whole-cell and lipid-extracted S-AMDD were estimated using a modified batch-culture in vitro fermentation system with total gas capture using lactating dairy cattle as rumen fluid donors. In vitro monogastric DPD and two-phase GPD were measured by i ncubation of whole-cell and lipid-extracted S-AMDD samples in porcine pepsin and porcine pancreatin, containing amylase, lipase and protease enzyme solutions. Results: Simulated ruminal fermentations using lactating dairy cattle as rumen fluid donors in dicate that both whole-cell and lipid-extracted S-AMDD have excellent potential for use in ruminant animal feeds. Dietary inclusion of whole-cell S-AMDD at 50% forage protein replacement (equivalent to 20% of the total diet) or lipid-extracted S-AMDD at 100% forage protein replacemen...