Identification of factors that limit plant cell wall digestion of forages and the development of enzymatic approaches that improve hydrolysis could play a key role in improving the production efficiency of ruminants fed these feeds. Enzyme fingerprinting and FTIR analysis of barley silage and total-tract indigestible fiber residue (TIFR) in heifers’ feces were used to identify cell wall components resistant to digestion. The results identified acetyl xylan esterases as key to enhance ruminal digestion, and that polysaccharide-lignin complexes are principal components in TIFR. Enzyme pre-treatment to enhance glucose yield from hydrolyses of barley straw and alfalfa hay by mixed rumen enzymes (MRE) was subsequently developed. The enzymes for pre-hydrolysis were recombinant acetyl xylan esterases (AXE16B_ASPNG and AXE16A_ASPNG), polygalacturonase (PGA28A_ASPNG), α-arabinofuranosidase (ABF54B_ASPNG) all from Aspergillus niger, feruloyl esterase (FAE1a) from Anaeromyces mucronatus (expressed in E. coli), endoglucanase GH7 (EGL7A_THITE) from Thielavia terrestris (produced in A. niger). Lyophilized cell-free rumen fluid from cows fed forage diet was used as source of MRE. The study was conducted using micro assays in combination with simplex lattice mixture models that were designed using Design of Experiment statistical software. Fungal hemicellulases and auxiliary enzymes initiated degradation of structural polysaccharides upon application and improved the in vitro saccharification of alfalfa and barley straw by MRE. The analysis model predicted 75% (SEM: 2.8%) higher relative glucose yield from alfalfa pretreated with PGA28A_ASPNG and ABF54B_ASPNG in 1:1 ratio. Whereas, prehydrolysis of barley straw with a mixture of 50% EGL7A_THITE and 50% FAE1a increased glucose release by 100% (SEM: 1.8%) upon incubation with MRE. The results showed that microassays in combination with simplex lattice statistical experimental design can be used to predict effective enzyme pretreatments that can enhance plant cell wall digestion by MRE, and strengthen the rationale of developing specific enzyme pretreatments for forages, depending on their structure and composition.