The purpose of this study was to evaluate the effect of allogenic leukocyte-reduced platelet-rich plasma on human tenocytes after treatment with prednisolone and to develop a standardization of its application for clinical practice. A leukocyte-reduced PRP was produced using the Arthrex Double Syringe (Arthrex, Inc., Naples, FL, USA), in a modified single-spin separation method. Human tenocytes were isolated from discarded rotator cuff segments. Tenocytes were cultured in the presence of PRP and prednisolone, both alone and in combination. Control samples were treated in media containing 2% FCS for 72h. After 72h of incubation, cell cycle kinetics of tenocytes were analyzed to assess proliferation. Incubation of the tenocytes with PRP alone for 48h led to high proliferation rate (10% PRP, 28.0±10.5%; 20% PRP, 40.9±3.3%). Incubation in the presence of prednisolone led to a significant decrease of the proliferation rate (5.2±3.1%; p<0.05). Treatment with PRP for 48h significantly increased the proliferation of tenocytes in a dose-dependent manner (10% PRP, 28.0±10.5%; 20% PRP, 40.9±3.3%; p<0.05). The presence of prednisolone resulted in a decreased tenocyte proliferation (5.2±3.1%; p<0.05), whereas addition of PRP for 24 and 48h after prednisolone exposure did not show any compensating effect independent of PRPs concentration (10% PRP, 3.7±3.0%; 20% PRP, 2.5±2.5%). However, a significantly increased cell proliferation of tenocytes was evident when PRP was applied 24 h after prednisolone incubation for 48h (31.0±3.4 and 34.3±4.7%). The use of leukocyte-reduced PRP stimulates the proliferation of tenocytes and antagonizes the negative effect of prednisolone 24h after treatment. Addition of PRP 48h after treatment with prednisolone has no positive effect on the proliferation rate of tenocytes.