Blood sample processing and handling can have a significant impact on the stability and levels of proteins measured in biomarker studies. Such pre-analytical variability needs to be well understood in the context of the different proteomics platforms available for biomarker discovery and validation. In the present study we evaluated different types of blood collection tubes including the BD P100 tube containing protease inhibitors as well as CTAD tubes, which prevent platelet activation. We studied the effect of different processing protocols as well as delays in tube processing on the levels of 55 mid and high abundance plasma proteins using novel multiple-reaction monitoring-mass spectrometry (MRM-MS) assays as well as 27 low abundance cytokines using a commercially available multiplexed bead-based immunoassay. The use of P100 tubes containing protease inhibitors only conferred proteolytic protection for 4 cytokines and only one MRM-MS-measured peptide. Mid and high abundance proteins measured by MRM are highly stable in plasma left unprocessed for up to six hours although platelet activation can also impact the levels of these proteins. The levels of cytokines were elevated when tubes were centrifuged at cold temperature, while low levels were detected when samples were collected in CTAD tubes. Delays in centrifugation also had an impact on the levels of cytokines measured depending on the type of collection tube used. Our findings can help in the development of guidelines for blood collection and processing for proteomic biomarker studies.