One of the most commonly faced clinical problems is dentinal hypersensitivity (DH).It's a "enigma" that's "often met yet seldom comprehended." It is defined as a sensation of discomfort caused by exposed dentine in response to heat, chemical, tactile, or osmotic stimulation. It appears to be a common ailment, with estimates ranging from 4% to 74 percent of the population.The study aimed in evaluating the efficacy of sodium fluoride varnish, dentine bonding agent and diode laser in treating dentine hypersensitivity in vitro and in vivo.The research was split into two parts: in vitro and in vivo. In the in vitro study, 40 anterior teeth were extracted and separated into four groups: control, group A (fluoride varnish), group B (dentine bonding agent), and group C (laser), all of which were studied under a scanning electron microscope. For the in vivo part 30 patients aged 20-50 years with the chief complaint of sensitivity to hot and cold were selected from the outpatient department (OPD) of Seema Dental college and Hospital. The patients were divided into 3 groups, group A(fluoride varnish), group B (dentine bonding agent) group C (laser).At the end of 3 months, there was a statistically significant difference seen in mean VAS and VRS scores between Group A, Group B, and Group C; additionally, the mean of dentinal tubules in the SEM study (In vitro) was seen to be significantly higher in the Control group compared to Group A and Group B, and significantly higher than Group C.According to the findings of this study, all three treatment methods, sodium fluoride varnish, dentine bonding agent and laser are efficient in reducing dentine hypersensitivity in both in vivo and vitro studies, with laser showing better outcomes.