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The purpose of this study was to use near-infrared (NIR) transmission spectroscopic technique to determine clindamycin plasma concentration after oral administration of clindamycin loaded GMO-alginate microspheres using rabbits as animal models. Lyophilized clindamycin–plasma standard samples at a concentration range of 0.001–10 μg/ml were prepared and analyzed by NIR and HPLC as a reference method. NIR calibration model was developed with partial least square (PLS) regression analysis. Then, a single dose in-vivo evaluation was carried out and clindamycin–plasma concentration was estimated by NIR. Over 24 h time period, the pharmacokinetic parameters of clindamycin were calculated for the clindamycin loaded GMO-alginate microspheres (F3) and alginate microspheres (F2), and compared with the plain drug (F1). PLS calibration model with 7-principal components (PC), and 8000–9200 cm −1 spectral range shows a good correlation between HPLC and NIR values with root mean square error of cross validation (RMSECV), root mean square error of prediction (RMSEP), and calibration coefficient ( R 2 ) values of 0.245, 1.164, and 0.9753, respectively, which suggests that NIR transmission technique can be used for drug-plasma analysis without any extraction procedure. F3 microspheres exhibited controlled and prolonged absorption T max of 4.0 vs. 1.0 and 0.5 h; C max of 2.37 ± 0.3 vs. 3.81 ± 0.8 and 5.43 ± 0.7 μg/ml for F2 and F1, respectively. These results suggest that the combination of GMO and alginate (1:4 w/w) could be successfully employed for once daily clindamycin microspheres formulation which confirmed by low C max and high T max values. [ABSTRACT FROM AUTHOR]