Honey is a prime biogenic compound produced by honeybees (Apis mellifera, L.) through the mixing of the nectar from various flowers and a plethora of enzymatic reactions inside their honey sacs. Physicochemical characterization and identification of bioactive compounds with nutritional properties present in honey hold significant importance in the fields of agriculture, microbiology, immunology, and pharmaceuticals. Several health-protective benefits of honey are well-documented, including anti-inflammatory, antimicrobial, and antioxidant, along with cardioprotective, hepatoprotective, and neuroprotective properties. Melissopalynology studies of honey provide a multitude of information regarding botanical and geographical origin. The traditional and high-throughput technologies including spectroscopic and chromatographic analysis of honey provide essential details comprising various parameters, which include identification of sugars, amino acids, organic acids, enzymes, vitamins, and phenolic compounds, detection and quantification of bioactive compounds, presence of pollen, pesticides, heavy metals, and environmental pollutants. All these sets of valuable information together could fetch the analytical underpinning with a comprehensive assessment of the chemical composition and quality of honey. DNA metabarcoding and next generation sequencing (NGS) analysis of honey provide significant botanical information, from detection of the floral origin to plant taxa identification. Additionally, entomological details are also to be taken into consideration, which can be efficacious for marketable value assessment of honey corresponding to public health benefits and pricing. These analytical and biochemical techniques elucidate instrumental advantages and application of comprehensive analysis of honey.