Pneumocystis pneumonia (PCP) is an opportunistic infection that occurs in humans and other mammals with debilitated immune systems. These infections are caused by fungi in the genus Pneumocystis, which are not susceptible to standard antifungal agents. Despite decades of research and drug development, the primary treatment and prophylaxis for PCP remains a combination of trimethoprim (TMP) and sulfamethoxazole (SMX) that targets two enzymes in folic acid biosynthesis, dihydrofolate reductase (DHFR) and dihydropteroate synthase (DHPS), respectively. There is growing evidence of emerging resistance by (the species that infects humans) to TMP-SMX associated with mutations in the targeted enzymes. In the present study, we report the development of an accurate quantitative model to predict changes in the binding affinity of inhibitors (, ) to the mutated proteins. The model is based on evolutionary information and amino acid covariance analysis. Predicted changes in binding affinity upon mutations highly correlate with the experimentally measured data. While trained on DHFR/TMP data, the model shows similar or better performance when evaluated on the resistance data for a different inhibitor of PjDFHR, another drug/target pair (PjDHPS/SMX) and another organism ( DHFR/TMP). Therefore, we anticipate that the developed prediction model will be useful in the evaluation of possible resistance of the newly sequenced variants of the pathogen and can be extended to other drug targets and organisms.