Liquid chromatography (LC) coupled to hybrid quadrupole time-of-flight (QTOF) mass spectrometry (MS) is a useful analytical tool in the elucidation and confirmation of transformation products (TPs)/metabolites of pesticides with a wide range of polarity, in both environmental and biological samples. Firstly, the versatility of LC allows the determination of very distinct TPs/metabolites as chromatographic conditions can be easily changed and optimized depending on the analytical problem. Secondly, the mass accuracy provided by the TOF analyser allows the assignment of a highly probable empirical formula for each compound and the differentiation between nominal isobaric compounds. Finally, the possibility of performing MS/MS spectra with accurate mass measurements can been used for the final characterization of the TPs/metabolites detected and for the differentiation of isomeric compounds. In this study, the insecticide diazinon was used as model compound, and its photodegradation and metabolism have been investigated by LC-QTOF-MS. On one hand, environmental spiked water was irradiated with a mercury lamp for 9 days, sampling 3-mL aliquots approximately every 12 h. On the other hand, both in vitro and in vivo metabolism experiments were carried out with different substrate concentrations and incubation times. After centrifugation, and protein precipitation in the in vitro and in vivo studies, 50-microL aliquots of both environmental and biological samples were directly injected into the LC electrospray ionization QTOF system. The most important transformation processes were found to be hydrolysis of the ester moiety, hydroxylation in the aromatic ring or in one of the alkylic groups, oxidation of the sulfur atom on the P=S cleavage or a combination of these processes, with the highest number of compounds being found in the photodegradation study. Very polar compounds, such as diethyl phosphate and diethyl thiophosphate, were detected after direct injection of the aqueous sample, which was feasible owing to the characteristics of the LC. In MS mode, mass errors were below 3 mDa, leading to an empirical formula for each compound. MS/MS spectra with accurate mass were used for the final elucidation of the compounds detected.