Introduction: The biochemical activities of gingival crevicular fluid (GCF) suggest the various alterations that occur in the gingival tissue during the orthodontic tooth movement. Alkaline phosphatase (ALP) and Cathepsin-B (CAB) act as markers in bone metabolism. This longitudinal study was conducted to examine the activity of ALP and CAB in GCF. Materials and Method: A split mouth study was conducted using both left and right maxillary quadrants for 20 sites in 10 subjects. After 6 weeks of placement of archwire, the canine retraction was initiated bilaterally on the 0.019x0.025” stainless steel base archwire. The initial force of approximately 200 gm was applied by using NiTi closed coil spring. GCF was collected using calibrated micropipette from mesial and distal sites of maxillary canines at different time intervals: T0 (before extraction), T1 (one hour prior to retraction), T2 (after 1 day), T3 (after 7 days), T4 (after 14 days) and T5 (after 21 days). After collection, GCF was transferred to eppendorf tube and stored at -200C. Results: Both ALP and CAB achieved peak level after applying the force for 24 hours followed by a significant reduction at 1st, 2nd and 3rd week. Conclusion: The levels of both biomarkers achieved peak level after the first day of force application followed by a significant decrease at 1 week, 2 weeks and 3 weeks. After 3 weeks, it was reduced to the initial level i.e. before application of retraction force. CAB was shown to have higher levels as compared to ALP at all time intervals.