Insulin analogues and large bioactive peptides may be used by athletes to enhance performance and are banned by the World Anti‐Doping Agency (WADA). In addition to insulin analogues, the large peptides include a structurally diverse set of peptides including analogues of growth hormone releasing hormone (GHRH), insulin‐like growth factor‐1 (IGF‐1), and mechano‐growth factor (MGF). Detection of this class of peptides is difficult due to their absorptive losses and presence at very low concentrations in urine. In this report, a high throughput method is described that allows sensitive detection of four classes of large peptides in one assay. Sample extraction is performed by ultrafiltration to concentrate the urine followed by solid phase extraction in a 96‐well micro‐elution plate. Peptides in the urine samples are detected on a triple quadrupole mass spectrometer coupled to standard flow liquid chromatography. The method was validated and evaluated for limit of detection, limit of identification, specificity, precision, carryover, recovery, matrix interference, and post‐extraction stability. The limit of detection for insulin analogues is between 5 and 25 pg/ml and between 5 and 50 pg/ml for the other peptide classes. Specificity was good with no detection of interfering peaks in blank urine samples. Carryover from a high concentration sample was not observed and the post‐extraction stability was between 77% and 107%. The method was able to detect insulin analogues in three diabetic urine samples. Increased screening for this class of peptides will improve detection and deterrence.