Creatine has been quantified in various tissues by a range of methodologies. This paper reports on the development and validation of a simplified HPLC assay to determine plasma creatine, plasma protein binding of creatine, creatine in microdialysate and creatine in over-the-counter products. An isocratic, reversed-phase (C 1 8 ) HPLC assay, using potassium phosphate monobasic (pH 4) as a mobile phase, was validated in human plasma and microdialysis perfusion fluid (normal saline). The lower limit of quantification for the assay was 1 mg l - 1 in saline and 5 mg l - 1 in plasma. The RSD was below 6% and accuracy was below 12% in both matrices. Protein binding in human plasma was found to be negligible (<10%). Over-the-counter creatine monohydrate products tested contained 100% creatine monohydrate. This assay was found to be suitable for pharmacokinetic studies and the assessment of plasma creatine and skeletal muscle microdialysate.