The objective was to evaluate protein expression and post-translation modifications in mammary and muscle tissues of cows supplemented with 2 AA groups. Four lactating cows were assigned to treatments of saline (CON), methionine plus lysine plus histidine (MKH), isoleucine plus leucine (IL), or MKH plus IL in a 4 × 4 Latin square. Milk protein yield increased with MKH and IL independently with the MKH⁺IL treatment yielding a 9.6% increase versus CON. Mammary and muscle proteomes were assessed for total and phosphorylated abundance and the respective ratio using proteomics. Total and site-specific phosphorylated abundances of mammalian target of rapamycin (mTOR), ribosomal protein S6 kinase (S6K1), ribosomal protein S6 (rpS6), and eukaryotic initiation factor 2a (eIF2a) were assessed by western immunoblotting. The treatments both independently affected the phosphorylation ratio of mTOR pathway proteins in the udder as indicated by multivariate analyses. For the udder proteome, there was a tendency for a treatment interaction on total and phosphorylated abundance as well as the phosphorylation ratio. Univariate proteomic analysis revealed an enhanced phosphorylation ratio of mitogen-activated protein kinase 1 (MAP2K1) by MKH and IL and insulin receptor substrate 1 (IRS1) by MKH treatment only which may indicate negative mTOR feedback. Western immunoblotting indicated decreased total and phosphorylated abundance of S6K1 as well as phosphorylation ratio for the MKH infusion. There was a positive treatment interaction for total and phosphorylated abundance of rpS6, S6K1, and eIF2a as well as the phosphorylation ratio of rpS6. No significant changes were observed in muscle. Results indicate that supplementing MKH or IL affects protein expression and phosphorylation ratio of many udder proteins. However, the changes in mTOR signaling proteins were generally opposite of expectations. The latter appears to be the result of negative feedback arising from infusion of MKH and IL alone and when infused in combination, less negative feedback and positive treatment interaction on mTORC1 targets.