Defects in the canonical PI3K/Akt signalling pathway are thought to contribute to lipid induced insulin resistance. Much of the work done to investigate insulin signalling pathways is carried out in cell lines such as 3T3-L1 adipocytes, C2C12 and L6 myocytes. These cell lines however, may not reflect the complexities of tissue physiology in vivo. We have used acute insulin infusion of rats to investigate phospho-proteomic changes in muscle by tandem mass spectrometry. Male Wistar rats were fed either chow or a high fat diet (HFD) (45% calories as fat). After 3 weeks of diet rats were implanted with cannulas and one week later rats were infused with saline or 0.5U/kg/h of insulin. Muscle was taken at 5 and 10 minutes after the start of insulin infusion. Red quadriceps tissue was processed and multiplexed by tandem mass tag labelling with internal controls over each run. Phosphopeptide enrichment was achieved by TiO2 and metal ion affinity chromatography (IMAC). Peptides were fractionated by hydrophilic interaction liquid chromatography and fractions scrutinized by electrospray ionization MS/MS through the Orbitrap platform. Data were processed through MaxQuant. There was a clear effect of insulin on the phosphorylation of known insulin regulated serine/threonine kinases such as Akt at S474 (insulin stimulated 1.7 fold over basal, p=0.0006). 203 phosphosites had significantly reduced phosphorylation (<0.8 fold) in HFD muscle compared to chow after insulin stimulation including phosphosites on 11 proteins in the insulin signalling cascade including glycogen synthase, IRS1 and Akt. Other putative insulin regulated phosphosites on TBC1D4 and GSK3β showed no difference in phosphorylation between chow and HFD muscle. 14 proteins involved in glucose metabolism also showed differential phosphorylation between insulin stimulated chow and HFD muscle. These data suggest that in muscle, particular nodes of the PI3K/Akt pathway are susceptible to lipid induced insulin resistance rather than reduced phosphorylation over the whole pathway.