The measurement of human insulin and its synthetic analogues in biological matrices has become increasingly important not only in clinical fields but also in doping control. The use of insulin and its analogues have been included in the list of prohibited substances published by the World Anti-Doping Agency (WADA). This study describes a qualitative method for detection of insulin analogues (lispro, aspart, glulisine, glargine, degludec, detemir) in human urine. The sample preparation consists of a preconcentration step using ultrafiltration followed by an immunoaffinity extraction with an antibody precoated ELISA plate. The obtained extracts are analyzed by conventional high-performance liquid chromatography–electrospray tandem mass spectrometry (LC-ESI–MS/MS). The limits of detection range between 10 pg/ml and 150 pg/ml. The applicability of the method was proven by the analysis of real urine samples obtained from diabetic patients treated with synthetic insulin analogues.