Parathyroid Hormone-like Hormone (PTHLH) exerts relevant roles in the progression and dissemination of several tumours. However, factors influencing its production and secretion have not been fully characterized. The main limitation is the lack of specific, sensitive and widely available techniques to detect and quantify PTHLH. We have developed a Lateral Flow Immunoassay using gold nanoparticles label for the fast and easy detection of PTHLH in lysates and culture media of three human cell lines (HaCaT, LA-N-1, SK-N-AS). Levels in culture media and lysates ranged from 11 to 20 ng/mL and 0.66 to 0.87 µg/mL respectively. Results for HaCaT are in agreement to the previously reported, whereas LA-N-1 and SK-N-AS have been evaluated for the first time. The system also exhibits good performance in human serum samples. This methodology represents a helpful tool for future in-vitro and invivo studies of mechanisms involved in PTHLH production as well as for diagnostics.