Insulin stimulates milk protein synthesis through repression of tuberous sclerosis protein complex and recruitment of the mechanistic target of rapamycin complex 1 (mTORC1) to the lysosomal membrane, where mTORC1 can be activated by AA. We analyzed the necessity of insulin signaling for essential AA (EAA) stimulation of mTORC1 substrates in mammary epithelial cells. MAC-T cells (n = 3) were serum and insulin starved overnight and further starved for EAA for 4 h before incubating for 1 h in Dulbecco’s modified Eagle’s medium supplemented with insulin (100 nM) or vehicle, and 0, 0.05, 0.1,0.3, 1.0, and 3.0 mM of EAA at the profile of casein. Nonessential AA were maintained at 1.2 mM. Intracellular proteins were isolated and analyzed by Western blotting for total and phosphorylated forms of the mTORC1 substrate and protein synthesis regulator S6 kinase 1 (Thr 389), its downstream substrate ribosomal protein S6 (Ser 240/244), and the mTORC1 substrate and autophagy initiator factor ULK1 (Ser 757). Amino acid dose response parameters (linear and quadratic) were estimated for the phosphorylated to total ratio of each of the above-mentioned proteins, using the linear model and confidence interval functions in RStudio. Amino acid response parameters were estimated independently in the presence and absence of insulin. Intercepts were higher in the presence of insulin, from 70% for ULK1 to 10-fold for S6K1, indicating an AA-independent effect of insulin on mTORC1 activity. On the other hand, essential AA significantly stimulated mTORC1 activity only in the presence of insulin, where linear and quadratic parameters for the 3 target proteins were significantly different than zero (P < 0.01). In the absence of insulin, linear and quadratic parameter estimates were no different than zero (P > 0.05), but significantly different than insulin-treated parameter (P < 0.05). Our results indicate that EAA have insulin-dependent saturable effects on mTORC1 activity within physiological levels, suggesting that disruption of insulin signaling would negatively affect milk protein synthesis.