An innovative method for the determination of pesticides of different uses and from different chemical families and/or their known metabolites in human urine was developed. The protocol includes an enzymatic hydrolysis followed by an off-line solid phase extraction (SPE) step and then a targeted quantitative analysis by ultra-high performance liquid chromatography coupled with hybrid quadrupole time-of-flight mass spectrometry (UHPLC/QTOFMS). Still rarely used for this purpose, this technique nevertheless offers excellent quantitative performance with high selectivity, but requires two separate injections for the same sample, one in the negative electrospray ionization mode (ESI-), and the other in the positive mode (ESI+). Two analytical methods were thus developed to cover 40 substances corresponding to 23 different pesticides plus nicotine and cotinine. The first method allows the determination of 15 biomarkers (SPE on a weak anion exchange polymeric support; ESI- mode) and the second 27 (SPE on a reverse phase polymeric support; ESI+ mode) with detection limits for a 2-mL urine sample varying from 0.02 to 25 μg.L−1. Replicate analysis of three pools of urine non-spiked and spiked at an intermediate level on about 20 different days showed most often excellent performance in terms of inter-day precision (<30%) and accuracy (between 80 and 125%), confirming that the UHPLC/QTOFMS coupling is perfectly adapted to a targeted quantitative analysis. The proposed methods were then applied to fifteen real urine samples, demonstrating the presence of pesticides, predominantly in metabolized form, in the urine of French pregnant women, and the capabilities of the proposed methods to detect them. They were finally applied to a study of the impact of thawing/freezing cycles on the conservation of the target substances, showing for the first time to our knowledge that the thawing/freezing cycles and the composition of urine samples could have an influence on the stability of some of the target substances.