<b><i>Introduction:</i></b> The use of novel kidney injury biomarkers has been shown to improve diagnostic assessment and prognostic prediction in various populations with acute kidney injury (AKI), but their use in a standard clinical practice have been rarely reported. <b><i>Methods:</i></b> We reported the clinical implementation of neutrophil gelatinase-associated lipocalin (NGAL) measurement for routine AKI diagnostic workup of patients receiving nephrology consultation in a tertiary academic centre. Specific focus was made on the diagnostic performance to discriminate functional (“pre-renal”) from intra-renal AKI and to predict AKI progression. <b><i>Results:</i></b> Forty-five urine NGAL (uNGAL) and 25 plasma NGAL (pNGAL) samples in the first 50 consecutive patients were analysed. KDIGO Stage 1, 2, 3 AKI, and renal replacement therapy occurred in 10%, 40%, 50%, and 24% of cases, respectively. The uNGAL was lower in patients with transient AKI (<48 h) and no sign of urinary tract infections (37 [25–167] ng/mL) than sustained or progressive AKI (298 [74–1,308] ng/mL) (<i>p</i> = 0.016), while pNGAL did not discriminate transient (264 [100–373] ng/mL) from persistent AKI (415 [220–816] ng/mL) (<i>p</i> = 0.137). The median uNGAL level was 63 (35–1,123) ng/mL for functional/pre-renal AKI and 451 (177–1,315) ng/mL for intra-renal AKI (<i>p</i> = 0.043), while the pNGAL was 264 (114–468) and 415 (230–816) ng/mL (<i>p</i> = 0.235), respectively. <b><i>Conclusion:</i></b> NGAL, as part of the routine workup, is useful for diagnostic and prognostic assessment of new-onset AKI in clinical practice. Interpretation of an increased NGAL level should be clinically evaluated in its clinical context, particularly considering concomitant infection (urinary or systemic). Clinical adoption of emerging AKI biomarkers as diagnostic tests in clinical practice should be further encouraged.