New thermodynamic and functional studies have been recently conducted to evaluate the impact of amino acid substitutions on the Mitogen Activated Protein Kinases 1 and 3 (MAPK1/3). The Critical Assessment of Genome Interpretation (CAGI) data provider, at Sapienza University of Rome, measured the unfolding free energy and the enzymatic activity of a set of variants (MAPK challenge dataset). Thermodynamic measurements for the denaturant-induced equilibrium unfolding of the phosphorylated and unphosphorylated forms of the MAPKs were obtained by monitoring the far-UV circular dichroism and intrinsic fluorescence changes as a function of denaturant concentration. These values have been used to calculate the change in unfolding free energy between the variant and wild-type proteins at zero concentration of denaturant (ΔΔGH2O). The enzymatic activity of the phosphorylated MAPKs variants was also measured using Chelation-Enhanced Fluorescence to monitor the phosphorylation of a peptide substrate. The MAPK challenge dataset, composed of a total of 23 single amino acid substitutions (11 and 12 for MAPK1 and MAPK3, respectively), was used to assess the effectiveness of the computational methods in predicting the ΔΔGH2O values, associated with the variants, and categorize them as destabilizing and not destabilizing. The data on the enzymatic activity of the MAPKs mutants were used to assess the performance of the methods for predicting the functional impact of the variants. For the sixth edition of CAGI, thirteen independent research groups from four continents (Asia, Australia, Europe and North America) submitted > 80 sets of predictions, obtained from different approaches. In this manuscript, we summarized the results of our assessment to highlight the possible limitations of the available algorithms.