Abstract A quantitative ratiometric method based on laser scanning confocal microscopic imaging was developed to create a pixel-by-pixel neutral microclimate pH (5.8–8.0) map inside poly(lactic-co-glycolic acid) (PLGA) microspheres. The method was accurate to within ±0.2 pH units. PLGA microspheres with encapsulated bovine serum albumin and the pH-sensitive dye, SNARF-1® dextran (MW=10 kDa), were prepared by a water-in-oil-in-water solvent evaporation method. The imaging method was used to monitor the pH changes in PLGA microspheres with or without co-encapsulation of acid-neutralizing MgCO 3, and for three different microsphere sizes (2.0±0.3, 40±10 and 137±32 μm) during incubation under physiological conditions. The pH of microspheres was significantly higher with co-encapsulated base compared to base-free microspheres, whose microclimate pH was predominantly below the dye detection limit of pH=5.8. The small microspheres displayed a more neutral pH during incubation probably due to their much shorter diffusion path for polymer-dissolved acids compared with medium- and large-size microspheres. The pH mapping technique discussed here may be useful in routine formulation development and for elucidation of mechanisms of microclimate pH development and polymer erosion.