Pollution of surface and groundwater by nitrate (NO3–) is one of the most serious environmental problems worldwide. The crucial isotopes for the sources and dynamics of nitrogen (N) in the aquatic environment are the nitrogen and oxygen isotope data of NO3-. The stable isotope of nitrogen (δ15N) in dissolved nitrate has been most commonly used to estimate NO3- sources in groundwater. However, measurement of the stable nitrogen isotope alone does not provide a definitive determination of NO3– origin, so the stable oxygen isotope (δ18O) must be used, i.e., a dual isotope approach. The analysis of NO3- for both δ15N and δ18O has been made possible by the development of various laboratory methods for sample preparation, each with its own advantages and disadvantages. Current methods such as the cadmium reduction method (Azide method) or the bacterial denitrification method require toxic chemicals or anaerobic bacterial cultures for NO3- reduction to N2O gas. In 2019, a new sample preparation method was developed (ALTABET et al., 2019), which is a simple one-step conversion method that utilizes Titanium( III) chloride reagent to reduce NO3– to N2O within septum sample vials: 2NO3 – (aq) + 8Ti+3 + 10H+ → N2O(g) +8Ti+4 + 5H2O A single sample preparation takes only a few minutes, followed by a 24-h reaction that generates N2O headspace gas for δ15N and δ18O analysis by IRMS or laser spectrometer. Briefly, Ti(III) chloride is preconditioned with zinc metal powder about 30 minutes before sample preparation to ensure efficiency by removing Ti(IV). The volume ratio of sample to reagent for groundwater samples is 10:1, 20:1, or 40:1. We found that higher amounts of Ti(III) reagent (10:1 and 20:1 ratios) gave slightly more accurate δ18O values. The δ15N values were most inaccurate at ratios of 10:1 and 40:1, but were more accurate at a 20:1 ratio of sample to reagent. Therefore, a 20:1 ratio is considered practical for further measurements. To ensure consistent N2O yields and 15N and 18O results, the N concentrations in each vial must be identical for all samples, laboratory controls, and standards. This is achieved by adding an appropriate volume of sample, degassed deionized water, 10% hydrochloric acid, and the preconditioned Ti(III) chloride reagent. The δ15N and δ18O isotope analyses were performed using the Isotopic N2O Laser Analyzer (GLA451-N2OI3), which was acquired as part of the CRO7002 project “Using Nitrogen and Oxygen Stable Isotopes in the Determination of Nitrate Origin in the Unsaturated and Saturated Zone of the Velika Gorica Wellfield”, funded by the International Atomic Energy Agency (IAEA). The described method is very competitive (simple, faster and more cost-effective) compared to the existing methods currently used in most laboratories worldwide, and its implementation at the Faculty of Mining, Geology and Petroleum Engineering (University of Zagreb) enabled the development of new research on the estimation of the origin of nitrate in groundwater.