A series of novel 2-(substitutedphenyl)-5-methyl-3-(phenylamino)-1,3-thiazolidin-4-ones (4a-g) were synthesized and structurally confirmed by elemental analysis, IR, 1H NMR and MS spectral data. All the synthesized 1,3- thiazolidin-4-one analogues (4a-g) at various concentrations (10, 20, 50, 100 and 200 mcg/ml) have been evaluatedfor in vitro cytotoxicity against Dalton’s lymphoma ascites (DLA) cancer cell line by trypan blue exclusion method, in comparison with standard drug doxorubicin hydrochloride. Out of these seven compounds, three compounds (2- (2,4-dichlorophenyl)-5-methyl-3-(phenylamino)-1,3-thiazolidin-4-one (4c), 2-(2,3-dichlorophenyl)-5-methyl-3- (phenylamino)-1,3-thiazolidin-4-one (4b) and 2-(4-bromophenyl)-5-methyl-3-(phenylamino)-1,3-thiazolidin-4-one (4d)) inhibited 70%, 68% and 61% DLA tumor cells at 100 mcg/ml concentration, whereas standard drug doxorubicin exhibit 100% DLA inhibition at a concentration of 100 mcg/ml. From the above study, compound 4b, compound 4c and compound 4d which showed better results (> 60% inhibition) at lowest concentration were further selected for screening in vivo anticancer activity against Dalton’s lymphoma ascites (DLA) cancer cell line at the dose of 50 mg/kg body weight/i.p. in comparison with 5-fluorouracil (20 mg/kg body weight/i.p.) by determining different parameters like body weight analysis, packed cell volume, viable tumor cell count, increase in life span (%), followed by hematological profiles [red blood cell (RBC), white blood cell (WBC), hemoglobin (Hb) and platelet count] and serum biochemical parameters [aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), total cholesterol (TC) and triglycerides (TG)] of DLA bearing mice. In the in vivo anticancer evaluation, among three compounds screened, compound 4c emerged as more potent inhibitor of DLA with an increase in life span (ILS) of 72.25%, whereas standard drug 5-fluorouracil exhibit ILS of 92.13%. The in vivo anticancer experimental results indicated that, compound 4c (p < 0.05) and 5-fluorouracil (p < 0.01) showed significant decrease in body weight gain, packed cell volume, viable tumor cell count and increased the life span of DLA tumor bearing mice, followed by hematological and serum biochemical profiles were significantly restored to normal levels in compound 4c (p < 0.05) and 5-Fluorouracil (p < 0.01) treated groups as compared to DLA control mice.