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Diazinon poisoning is an important issue in occupational, clinical, and forensic toxicology. While sensitive and specific enough to analyse diazinon in biological samples, current methods are time-consuming and too expensive for routine analysis. The aim of this study was therefore to design and validate a simple dispersive liquid-liquid microextraction (DLLME) for the preparation of urine samples to be analysed for diazinon with high performance liquid chromatography with diode-array detector (HPLC-DAD) to establish diazinon exposure and poisoning. To do that, we first identified critical parameters (type and volume of extraction and disperser solvents, pH, surfactant, and salt concentrations) in preliminary experiments and then used central composite design to determine the best experimental conditions for DLLME-HPLC-DAD. For DLLME they were 800 μL of methanol (disperser solvent) and 310 μL of toluene (extraction solvent) injected to the urine sample rapidly via a syringe. The sample was injected into a HPLC-DAD (C18 column, 250×4.6 mm, 5 μm), and the mobile phase was a mixture of acetonitrile and buffer (63:37 v/v, pH 3.2; flow rate: 1 mL/ min). Standard calibration curves for diazinon were linear with the concentration range of 0.5–4 μg/mL, yielding a regression equation Y=0.254X+0.006 with a correlation coefficient of 0.993. The limit of detection and limit of quantification for diazinon were 0.15 μg/mL and 0.45 μg/mL, respectively. The proposed method was accurate, precise, sensitive, and linear over a wide range of diazinon concentrations in urine samples. This method can be employed for diazinon analysis in routine clinical and forensic toxicology settings., Trovanje diazinonom važan je problem za medicinu rada te kliničku i forenzičnu toksikološku praksu. Premda su postojeće metode njegova utvrđivanja u biološkim uzorcima dovoljno osjetljive i specifične, njihova je primjena za rutinske analize preskupa i dugotrajna. Zbog toga je cilj ovoga istraživanja bio razviti i validirati jednostavnu metodu disperzivne tekućinsko-tekućinske mikroekstrakcije (engl. dispersive liquid-liquid microextraction, krat. DLLME) kojom bi se uzorci ljudske mokraće pripremili za analizu diazinona tekućinskom kromatografijom visoke djelotvornosti s detektorom s nizom fotodioda (engl. high performance liquid chromatography with diode-array detector, krat. HPLC-DAD) radi utvrđivanja izloženosti i trovanja diazinonom. U tu smo svrhu u preliminarnim eksperimentima prvo odredili ključne parametre: vrstu i volumen ekstrakcijskoga i disperzijskoga otapala, pH, površinski aktivne tvari (surfaktanta) te koncentraciju soli. Zatim smo s pomoću eksperimentalnoga centralnoga kompozitnoga dizajna utvrdili optimalne eksperimentalne uvjete za DLLME-HPLC-DAD. Za DLLME oni su bili 800 μL metanola (kao disperzijskoga otapala) te 310 μL toluena (kao ekstrakcijskoga otapala) za brzu injekciju uzorka mokraće. Uzorak je injektiran u HPLC-DAD (5-mikrometarski analitički stupac C18, 250×4,6 mm), a mobilna je faza bila mješavina acetonitrila i pufera (63:37 v/v, pH 3.2; protok: 1 mL/min). Standardne su kalibracijske krivulje za diazinon bile linearne s rasponom koncentracija diazinona od 0,5 do 4 μg/mL, a regresijska jednadžba Y=0,254X+0,006 s koeficijentom korelacije 0,993. Granice detekcije odnosno kvantifikacije diazinona bile su 0,15 μg/mL odnosno 0,45 μg/mL. Ova se metoda pokazala točnom, preciznom, osjetljivom i linearnom u širokom rasponu koncentracija diazinona u uzorcima mokraće, stoga se može koristiti za njegovu rutinsku analizu u kliničkoj i forenzičnoj toksikološkoj praksi.