We performed total proteome and phosphoproteome analysis to characterize the role of non-mitotic kinase activity of CDK1 in insulin signaling through pharmacological inhibition and translational repression via siRNA-based knockdown. Specifically, cultured myotubes were treated with RO-3306, a selective ATP competitive inhibitor to inhibit CDK1 activity, or transfected with a CDK1 targeting siRNA pool to decrease CDK1 expression.