Expanding interest in oxytocin, particularly the role of endogenous oxytocin in human social behavior, has created a pressing need for replication of results and verification of assay methods. In this study, we sought to replicate and extend previous results correlating plasma oxytocin with trust and trustworthy behavior. As a necessary first step, the two most commonly used commercial assays were compared in human plasma via the addition of a known quantity of exogenous oxytocin, with and without sample extraction. Plasma sample extraction was found to be critical in obtaining repeatable concentrations of oxytocin. In the subsequent trust experiment, twelve samples in duplicate, from each of 82 participants, were collected over approximately six hours during the performance of a Prisoner's Dilemma task paradigm that stressed human interpersonal trust. We found no significant relationship between plasma oxytocin concentrations and trusting or trustworthy behavior. In light of these findings, previous published work that used oxytocin immunoassays without sample extraction should be reexamined and future research exploring links between endogenous human oxytocin and trust or social behavior should proceed with careful consideration of methods and appropriate biofluids for analysis.