Photodynamic therapy (PDT) is a well-recognized treatment modality for oncological and non-oncological diseases. In this current research Rhodamine (Rh-640 perchlorate), a second generation photosensitizer, mediated photodynamic effects are evaluated. Different controlling parameters i.e., optimum intracellular drug concentration, light dose response on lower scale are investigated on in-vitro human Rhabdomyosarcoma cancer cell line. Diode laser 630 nm was used as a light source and three treatment arms were selected for photodynamic efficacy evaluation. An optimum incubation time of 3 hours was found for Rh-640 mediated exposure of RD culture. Effective intracellular drug accumulation was optimized for culture when administered with different concentrations (5, 10, 20, 30, 50, 70, and 100) µM. Photodynamic action of Rh-640 at different light doses (2, 5, 15) J/cm2 was evaluated. It was found that post incubation with optimized parameters Rhodamine 640 mediated photodynamic therapy showed light dose dependent cytotoxicity i.e., (85% (2 J/cm2), 80%( 5 J/cm2), 69%( 15 J/cm2). Current research work suggest the potential photodynamic action of Rh-640 and may be evaluated its efficacy on other cancer cell lines.