Phosphorylation is a dynamic, highly regulated post-translational modification that may be affected by variation in pre-analytical sample handling, hampering the translational value of phosphoproteomic-based data. Here, we investigate the temporal effect of delay in mononuclear cell isolation on acute myeloid leukemia (AML) phosphorylation profiles. We performed MS on immuno-precipitated phosphotyrosine (pY)-containing peptides isolated from AML samples after seven pre-defined delays before sample processing (direct processing, thirty minutes, one hour, two hours, three hours, four hours and 24 hours delay). Up to four hours, phosphorylation profiles showed limited variation. However, in samples processed with a delay of 24 hours, we observed significant change in phosphorylation profiles, with differential phosphorylation of 22 phosphopeptides (p < 0.01). This included increased phosphorylation of phosphopeptides of JNK and p38 kinases indicative of stress response activation. Based on these results, we conclude that processing of AML samples should be standardized at all times and should occur within four hours after sample collection.