This article provides a comprehensive overview of methods for detecting nitric oxide (NO) and its metabolites in biological samples. NO, a short-lived gaseous radical, plays crucial roles in various physiological processes such as blood pressure regulation, immune response, and neural communication. Accurate detection and quantification of NO are essential for understanding health and disease. The review highlights the limitations of traditional methods, such as the short physiological half-life of NO, and emphasizes the need for methods that can detect NO and its metabolites in multiple compartments of experimental animals. The article discusses the physiological chemistry of NO, including its production by nitric oxide synthases (NOS) and its oxidation to nitrite (NO₂⁻) and nitrate (NO₃⁻). It also covers the formation of S-nitrosothiols (RSNOs) and other NO-derived metabolites, which are important for understanding NO's biological functions. The importance of sample preparation in preserving NO and its metabolites is emphasized, with specific protocols for blood and tissue samples. Several detection methods are described, including colorimetric, fluorometric, and chemiluminescence-based approaches. Colorimetric methods, such as the Griess reaction, are widely used for their simplicity and sensitivity. Fluorometric methods, like the diaminonaphthalene (DAN) assay and diaminofluorescein-2 (DAF-2) assay, offer higher sensitivity and specificity. Chemiluminescence methods, such as the ozone-based chemiluminescent detector (CLD), are effective for detecting various NO derivatives, including RSNOs and nitrosyl heme compounds. The article also addresses the quantification of 3-nitrotyrosine (3NT), a post-translational modification of proteins and peptides, which is often used as a marker for nitrative stress. Various techniques, including immunohistochemistry, HPLC, and mass spectrometry (MS), are discussed for their specificity and sensitivity in quantifying 3NT. Finally, the article discusses the controversies and limitations associated with NO detection methods, emphasizing the importance of validating methods to ensure accurate quantification and interpretation of results. The review aims to provide a practical guide for researchers to choose the most suitable method for their specific needs.This article provides a comprehensive overview of methods for detecting nitric oxide (NO) and its metabolites in biological samples. NO, a short-lived gaseous radical, plays crucial roles in various physiological processes such as blood pressure regulation, immune response, and neural communication. Accurate detection and quantification of NO are essential for understanding health and disease. The review highlights the limitations of traditional methods, such as the short physiological half-life of NO, and emphasizes the need for methods that can detect NO and its metabolites in multiple compartments of experimental animals. The article discusses the physiological chemistry of NO, including its production by nitric oxide synthases (NOS) and its oxidation to nitrite (NO₂⁻) and nitrate (NO₃⁻). It also covers the formation of S-nitrosothiols (RSNOs) and other NO-derived metabolites, which are important for understanding NO's biological functions. The importance of sample preparation in preserving NO and its metabolites is emphasized, with specific protocols for blood and tissue samples. Several detection methods are described, including colorimetric, fluorometric, and chemiluminescence-based approaches. Colorimetric methods, such as the Griess reaction, are widely used for their simplicity and sensitivity. Fluorometric methods, like the diaminonaphthalene (DAN) assay and diaminofluorescein-2 (DAF-2) assay, offer higher sensitivity and specificity. Chemiluminescence methods, such as the ozone-based chemiluminescent detector (CLD), are effective for detecting various NO derivatives, including RSNOs and nitrosyl heme compounds. The article also addresses the quantification of 3-nitrotyrosine (3NT), a post-translational modification of proteins and peptides, which is often used as a marker for nitrative stress. Various techniques, including immunohistochemistry, HPLC, and mass spectrometry (MS), are discussed for their specificity and sensitivity in quantifying 3NT. Finally, the article discusses the controversies and limitations associated with NO detection methods, emphasizing the importance of validating methods to ensure accurate quantification and interpretation of results. The review aims to provide a practical guide for researchers to choose the most suitable method for their specific needs.